Synthesis of olefinic alcohols via enzymatic terminal hydroxylation

ABSTRACT

In certain aspects, the present invention provides methods for producing terminally hydroxylated alkenes and alkynes by contacting an unsaturated or saturated hydrocarbon substrate with a hydroxylase enzyme. Exemplary terminal hydroxylases useful for carrying out the methods of the invention exhibit strong selectivity towards one terminal carbon of a hydrocarbon substrate and include, but are not limited to, non-heme diiron alkane monooxygenases, cytochromes P450 (e.g., cytochromes P450 of the CYP52 and CYP153 family), as well as long chain alkane hydroxylases. In some embodiments, the terminally hydroxylated alkene or alkyne is further converted to a terminal alkenal. In certain embodiments, terminally hydroxylated alkenes and alkynes are useful as insect pheromones which modify insect behavior. In other embodiments, terminally hydroxylated alkenes and alkynes are useful intermediates for producing pheromones via acetylation or oxidation of the alcohol moiety.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a continuation of PCT/US2015/031219, filed May 15, 2015, which application claims priority to U.S. Provisional Application No. 61/994,662, filed May 16, 2014; U.S. Provisional Application No. 62/060,469, filed Oct. 6, 2014; U.S. Provisional Application No. 62/062,758, filed Oct. 10, 2014; U.S. Provisional Application No. 62/082,555, filed Nov. 20, 2014; U.S. Provisional Application No. 62/096,417, filed Dec. 23, 2014; and U.S. Provisional Application No. 62/096,429, filed Dec. 23, 2014; which applications are incorporated herein by reference in their entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

Certain aspects of this invention were made with government support under award number IIP-1448692, awarded by the National Science Foundation. The government may have certain rights in the invention.

BACKGROUND OF THE INVENTION

Insect pheromones can be used in a variety of insect control strategies that include mating disruption and attract-and-kill, as well as mass trapping. These strategies have proven to be effective, selective (e.g., they do not harm beneficial insects, such as bees and lady bugs), and safe (e.g., the compounds are generally biodegradable and do not accumulate in the food chain). Even the very stringent USDA Organic Program lists insect pheromones as one of the few synthetic organic compounds allowed in organic crop production, another important recognition of the high safety of these products. Accordingly, pheromones already form the basis of integrated pest management (IPM) practices in fruit production on the U.S. west coast, and their use in organic farming is growing worldwide.

Despite these advantages, pheromones are not widely used today because of the high cost of about $500 to $14,000 per kg of active ingredient (AI). Even though thousands of insect pheromones have been identified, less than about twenty insect pests worldwide are currently controlled using pheromone strategies, and only 0.05% of global agricultural land employs pheromones.

Lepidopteran pheromones, which are naturally occurring compounds, or identical or substantially similar synthetic compounds, are designated by an unbranched aliphatic chain (between 9 and 18 carbons) ending in an alcohol, aldehyde, or acetate functional group and containing up to 3 double bonds in the aliphatic backbone.

The present invention provides methods by which lepidopteran insect pheromones as well as structurally related compounds are prepared using synthetic strategies that are enabled by a biocatalytic step.

BRIEF SUMMARY OF THE INVENTION

In a first aspect, the invention provides a method for synthesizing an olefinic alcohol product that includes incubating an unsaturated hydrocarbon substrate with an enzyme capable of selectively hydroxylating one terminal carbon of the unsaturated hydrocarbon substrate to form an unsaturated hydrocarbon alcohol. In some embodiments, the unsaturated hydrocarbon alcohol is the olefinic alcohol product. In some embodiments, the method further includes converting the unsaturated hydrocarbon alcohol to the olefinic alcohol product.

In some embodiments, the unsaturated hydrocarbon substrate is an olefinic substrate. The olefinic substrate can be prepared via olefin metathesis and other routes including alkylation and reduction of alkynes, as well as Wittig-type reaction of aldehydes with phosphine reagents.

In some embodiments, the unsaturated hydrocarbon substrate is an alkyne. In some embodiments, the unsaturated hydrocarbon substrate is an alkenyl halide.

In a related aspect, the invention provides a method for synthesizing an olefinic alcohol product that includes incubating a saturated hydrocarbon substrate with an enzyme capable of selectively hydroxylating one terminal carbon of the saturated hydrocarbon substrate to form a saturated hydrocarbon alcohol, and converting the saturated hydrocarbon alcohol to the olefinic alcohol product.

In some embodiments, the saturated hydrocarbon substrate is an alkane substrate. In some embodiments, the method includes incubating the alkane substrate with an enzyme capable of selectively hydroxylating both terminal carbons of the alkane substrate to form a terminal diol. The terminal diol can be converted to the olefinic alcohol product in one or more subsequent steps.

In some embodiments, the saturated hydrocarbon substrate is an alkyl halide. In some embodiments, the method includes incubating the alkyl halide with an enzyme capable of selectively hydroxylating one terminal carbon of the alkyl halide to form a halogen-substituted alkanol. The halogen-substituted alkanol can be converted to the olefinic alcohol product in one or more subsequent steps.

In some embodiments, the saturated hydrocarbon substrate is a fatty acid. In some embodiments, the method includes incubating the fatty acid with an enzyme capable of selectively hydroxylating the terminal carbon of the fatty acid to form a terminal hydroxy fatty acid. The terminal hydroxy fatty acid can be converted to the olefinic alcohol product in one or more subsequent steps.

In some embodiments, the enzyme used in the methods of the invention is a non-heme diiron monooxygenase. In some embodiments, the enzyme is a long-chain alkane hydroxylase. In some embodiments, the enzyme is a cytochrome P450. In some embodiments, the cytochrome P450 is a member of the CYP52 or CYP153 family.

In certain embodiments, the olefinic alcohol product prepared according to the method of the invention is a pheromone. In particular embodiments, the pheromone is a lepidopteran insect pheromone.

In another aspect, the invention provides a whole cell catalyst comprising an enzyme capable of selectively hydroxylating one terminal carbon of an unsaturated or saturated hydrocarbon substrate. In some embodiments, the cell is a microbial cell. In some embodiments, the enzyme is selected from the group consisting of a non-heme diiron monooxygenase, a long-chain alkane hydroxylase, a cytochrome P450, and combinations thereof.

Other objects, features, and advantages of the present invention will be apparent to one of skill in the art from the following detailed description and figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the bioconversion reaction of (Z)-5-hexadecene using induced strain SPV048. (Z)-5-hexedecen-1-ol elutes at 11.5 min while (Z)-11-hexadecen-1-ol elutes at 11.6 min.

FIG. 2 shows a reaction scheme for hydroxylation of symmetric alkenes using the methods of the invention.

FIG. 3 shows a reaction scheme for hydroxylation of asymmetric alkenes using the methods of the invention.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The present invention provides a method of producing terminally oxyfunctionalized alkenes and alkynes. In certain aspects, the method includes contacting an unsaturated or saturated hydrocarbon substrate with a hydroxylase enzyme in the presence of molecular oxygen, reducing equivalents, and optionally redox partners under conditions sufficient to provide a terminally hydroxylated alkene or alkyne. The unsaturated or saturated hydrocarbon substrates can be prepared using the methods described herein. Relevant terminal hydroxylases useful for carrying out this method exhibit strong selectivity towards the terminal carbon of an alkyl chain and include, but are not limited to, non-heme diiron alkane monooxygenases, cytochromes P450 (e.g., cytochromes P450 of the CYP52 and CYP153 family), as well as long chain alkane hydroxylases. In certain embodiments, the terminally hydroxylated alkene or alkyne is further converted to a terminal alkenal. The terminal alkenal can be obtained by chemically or enzymatically oxidizing the terminally hydroxylated alkene or alkyne. Alcohol oxidases, alcohol dehydrogenases, and alpha-dioxygenases can be used for the enzymatic oxidation. In certain embodiments, terminally hydroxylated alkenes and alkynes are useful as insect pheromones which modify insect behavior. In some embodiments, terminally hydroxylated alkenes and alkynes are useful intermediates for producing pheromones via acetylation or oxidation of the alcohol moiety.

II. Definitions

The following definitions and abbreviations are to be used for the interpretation of the invention. The term “invention” or “present invention” as used herein is a non-limiting term and is not intended to refer to any single embodiment but encompasses all possible embodiments.

As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having, “contains,” “containing,” or any other variation thereof, are intended to cover a non-exclusive inclusion. A composition, mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive “or” and not to an exclusive “or.”

The terms “about” and “around,” as used herein to modify a numerical value, indicate a close range surrounding that explicit value. If “X” were the value, “about X” or “around X” would indicate a value from 0.9X to 1.1X, and more preferably, a value from 0.95X to 1.05X. Any reference to “about X” or “around X” specifically indicates at least the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, “about X” and “around X” are intended to teach and provide written description support for a claim limitation of, e.g., “0.98X.”

The terms “engineered enzyme” and “enzyme variant” include any enzyme comprising at least one amino acid mutation with respect to wild-type and also include any chimeric protein comprising recombined sequences or blocks of amino acids from two, three, or more different enzymes.

The terms “engineered heme enzyme” and “heme enzyme variant” include any heme-containing enzyme comprising at least one amino acid mutation with respect to wild-type and also include any chimeric protein comprising recombined sequences or blocks of amino acids from two, three, or more different heme-containing enzymes.

The terms “engineered cytochrome P450” and “cytochrome P450 variant” include any cytochrome P450 enzyme comprising at least one amino acid mutation with respect to wild-type and also include any chimeric protein comprising recombined sequences or blocks of amino acids from two, three, or more different cytochrome P450 enzymes.

The term “whole cell catalyst” includes microbial cells expressing hydroxylase enzymes, wherein the whole cell catalyst displays hydroxylation activity.

As used herein, the term “metathesis reaction” refers to a catalytic reaction which involves the interchange of alkylidene units (i.e., R₂C=units) among compounds containing one or more carbon-carbon double bonds (e.g., olefinic compounds) via the formation and cleavage of the carbon-carbon double bonds. Metathesis can occur between two like molecules (often referred to as self-metathesis) and/or between two different molecules (often referred to as cross-metathesis).

As used herein, the term “metathesis catalyst” refers to any catalyst or catalyst system that catalyzes a metathesis reaction. One of skill in the art will appreciate that a metathesis catalyst can participate in a metathesis reaction so as to increase the rate of the reaction, but is itself not consumed in the reaction.

As used herein, the term “metathesis product” refers to an olefin containing at least one double bond, the bond being formed via a metathesis reaction.

As used herein, the terms “microbial,” “microbial organism,” and “microorganism” include any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya. Therefore, the term is intended to encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea, and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. Also included are cell cultures of any species that can be cultured for the production of a chemical.

As used herein, the term “non-naturally occurring”, when used in reference to a microbial organism or enzyme activity of the invention, is intended to mean that the microbial organism or enzyme has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the microbial organism's genetic material. Such modifications include, for example, coding regions and functional fragments thereof, for heterologous, homologous, or both heterologous and homologous polypeptides for the referenced species. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon. Exemplary non-naturally occurring microbial organism or enzyme activity includes the hydroxylation activity described above.

As used herein, the term “exogenous” is intended to mean that the referenced molecule or the referenced activity is introduced into the host microbial organism. The term as it is used in reference to expression of an encoding nucleic acid refers to the introduction of the encoding nucleic acid in an expressible form into the microbial organism. When used in reference to a biosynthetic activity, the term refers to an activity that is introduced into the host reference organism.

The term “heterologous” as used herein with reference to molecules, and in particular enzymes and polynucleotides, indicates molecules that are expressed in an organism other than the organism from which they originated or are found in nature, independently of the level of expression that can be lower, equal or higher than the level of expression of the molecule in the native microorganism.

On the other hand, the terms “native” and/or “endogenous” as used herein with reference to molecules, and in particular enzymes and polynucleotides, indicate molecules that are expressed in the organism in which they originated or are found in nature, independently of the level of expression that can be lower equal or higher than the level of expression of the molecule in the native microorganism. It is to be understood that expression of native enzymes or polynucleotides may be modified in recombinant microorganisms.

The term “homolog,” as used herein with respect to an original enzyme or gene of a first family or species, refers to distinct enzymes or genes of a second family or species which are determined by functional, structural, or genomic analyses to be an enzyme or gene of the second family or species which corresponds to the original enzyme or gene of the first family or species. Homologs most often have functional, structural, or genomic similarities. Techniques are known by which homologs of an enzyme or gene can readily be cloned using genetic probes and PCR. Identity of cloned sequences as homologs can be confirmed using functional assays and/or by genomic mapping of the genes.

A protein has “homology” or is “homologous” to a second protein if the amino acid sequence encoded by a gene has a similar amino acid sequence to that of the second gene. Alternatively, a protein has homology to a second protein if the two proteins have “similar” amino acid sequences. Thus, the term “homologous proteins” is intended to mean that the two proteins have similar amino acid sequences. In certain instances, the homology between two proteins is indicative of its shared ancestry, related by evolution.

The terms “analog” and “analogous” include nucleic acid or protein sequences or protein structures that are related to one another in function only and are not from common descent or do not share a common ancestral sequence. Analogs may differ in sequence but may share a similar structure, due to convergent evolution. For example, two enzymes are analogs or analogous if the enzymes catalyze the same reaction of conversion of a substrate to a product, are unrelated in sequence, and irrespective of whether the two enzymes are related in structure.

As used herein, the term “alkane” refers to a straight or branched, saturated, aliphatic hydrocarbon having the number of carbon atoms indicated. The term “alkyl” refers to a straight or branched, saturated, aliphatic radical having the number of carbon atoms indicated. Alkyl can include any number of carbons, such as C₁₋₂, C₁₋₃, C₁₋₄, C₁₋₅, C₁₋₆, C₁₋₇, C₁₋₈, C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₃₋₄, C₃₋₅, C₃₋₆, C₄₋₅, C₄₋₆ and C₅₋₆. For example, C₁₋₆ alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc. Alkyl can refer to alkyl groups having up to 20 carbons atoms, such as, but not limited to heptyl, octyl, nonyl, decyl, etc. Alkanes and alkyl groups can be optionally substituted with one or more moieties selected from halo, alkenyl, and alkynyl.

As used herein, the term “alkene” refers to a straight chain or branched hydrocarbon having at least 2 carbon atoms and at least one double bond. A “terminal” alkene refers to an alkene wherein the double bond is between two carbon atoms at the end of the hydrocarbon chain (e.g., hex-1-ene). An “internal” alkene refers to an alkene wherein the double bond is between two carbon atoms that are not at the end of the hydrocarbon chain (e.g., (E)-hex-3-ene and (Z)-hex-3-ene). An “α,ω-alkenol” refers to a hydroxy-substituted terminal alkene having the formula (CH₂═CH)(CH₂)_(m)OH, wherein m is an integer ranging from 1-30, such as 2-18. The term “alkenyl” refers to a straight chain or branched hydrocarbon radical having at least 2 carbon atoms and at least one double bond. Alkenyl can include any number of carbons, such as C₂, C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₂₋₇, C₂₋₈, C₂₋₉, C₂₋₁₀, C₃, C₃₋₄, C₃₋₅, C₃₋₆, C₄, C₄₋₅, C₄₋₆, C₅, C₅₋₆, and C₆. Alkenyl groups can have any suitable number of double bonds, including, but not limited to, 1, 2, 3, 4, 5 or more. Examples of alkenyl groups include, but are not limited to, vinyl (ethenyl), propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl, or 1,3,5-hexatrienyl. Alkenes and alkenyl groups can be optionally substituted with one or more moieties selected from halo, alkyl, and alkynyl.

As used herein, the term “selective” refers to preferential reaction of one site on a chemical compound over another site on the compound. As a non-limiting example, selectively hydroxylating hept-3-ene (an asymmetric alkene) refers to preferentially hydroxylating one end of the hept-3-ene to form more hept-3-en-1-ol than hept-4-en-1-ol (or forming exclusively hept-3-en-1-ol without forming hept-4-en-1-ol). Selectively hydroxylating the other end of hept-3-ene would result in the formation of more hept-4-en-1-ol than hept-3-en-1-ol (or the exclusive formation of hept-4-en-1-ol without formation of hept-3-en-1-ol).

As used herein, the term “alkyne” refers to either a straight chain or branched hydrocarbon having at least 2 carbon atoms and at least one triple bond. A “terminal” alkyne refers to an alkyne wherein the triple bond is between two carbon atoms at the end of the hydrocarbon chain (e.g., hex-1-yne). An “internal” alkyne refers to an alkyne wherein the triple bond is between two carbon atoms that are not at the end of the hydrocarbon chain (e.g., hex-3-yne). The term “alkynyl” refers to either a straight chain or branched hydrocarbon radical having at least 2 carbon atoms and at least one triple bond. Alkynyl can include any number of carbons, such as C₂, C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₂₋₇, C₂₋₈, C₂₋₉, C₂₋₁₀, C₃, C₃₋₄, C₃₋₅, C₃₋₆, C₄, C₄₋₅, C₄₋₆, C₅, C₅₋₆, and C₆. Examples of alkynyl groups include, but are not limited to, acetylenyl, propynyl, 1-butynyl, 2-butynyl, isobutynyl, sec-butynyl, butadiynyl, 1-pentynyl, 2-pentynyl, isopentynyl, 1,3-pentadiynyl, 1,4-pentadiynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,3-hexadiynyl, 1,4-hexadiynyl, 1,5-hexadiynyl, 2,4-hexadiynyl, or 1,3,5-hexatriynyl. Alkynes and alkynyl groups can be optionally substituted with one or more moieties selected from halo, alkyl, and alkenyl.

As used herein, the term “aryl” refers to an aromatic carbon ring system having any suitable number of ring atoms and any suitable number of rings. Aryl groups can include any suitable number of carbon ring atoms, such as, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ring atoms, as well as from 6 to 10, 6 to 12, or 6 to 14 ring members. Aryl groups can be monocyclic, fused to form bicyclic or tricyclic groups, or linked by a bond to form a biaryl group. Representative aryl groups include phenyl, naphthyl and biphenyl. Other aryl groups include benzyl, having a methylene linking group. Some aryl groups have from 6 to 12 ring members, such as phenyl, naphthyl or biphenyl. Other aryl groups have from 6 to 10 ring members, such as phenyl or naphthyl. Some other aryl groups have 6 ring members, such as phenyl. Aryl groups can be optionally substituted with one or more moieties selected from halo, hydroxy, amino, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, and cyano.

As used herein, the terms “halo” and “halogen” refer to fluorine, chlorine, bromine and iodine.

As used herein, the term “carboxy” refers to a moiety —C(O)OH. The carboxy moiety can be ionized to form the carboxylate anion.

As used herein, the term “hydroxy” refers to a moiety —OH.

As used herein, the term “amino” refers to a moiety —NR₃, wherein each R group is H or alkyl. An amino moiety can be ionized to form the corresponding ammonium cation.

As used herein, the term “amido” refers to a moiety —NRC(O)R or —C(O)NR₂, wherein each R group is H or alkyl.

As used herein, the term “nitro” refers to the moiety —NO₂.

As used herein, the term “oxo” refers to an oxygen atom that is double-bonded to a compound (i.e., O═).

As used herein, the term “cyano” refers to the moiety —CN.

III. Description of the Embodiments

Traditionally, straight chain monoene alcohols, acetates, and aldehydes are synthesized via multi-step syntheses. Scheme 1 represents an example of such synthesis.

The present disclosure describes several methods for the synthesis of terminally oxyfunctionalized alkenes. Said methods are described in detail below and are generally applicable to the synthesis of various compounds, including but not limited to those shown in Table 1.

Some embodiments of the invention provide methods for synthesizing olefinic alcohol products wherein the olefinic alcohol product is a pheromone. In some embodiments, the olefinic alcohol product is selected from the alcohols in Table 1. Pheromones containing aldehyde functional groups can also be prepared using the olefinic alcohol products as intermediates. In such cases, the methods of the invention generally include oxidizing the olefinic alcohol product to form an olefinic aldehyde product. In some of these embodiments, the olefinic aldehyde product is selected from the aldehydes in Table 1.

Pheromones containing ester functional groups can also be prepared using the olefinic alcohol products as intermediates. In such cases, the methods of the invention generally include esterifying the olefinic alcohol product to form an olefinic ester product. In some embodiments, the olefinic ester product is an acetate ester. In some embodiments, the olefinic ester product is selected from the esters in Table 1.

TABLE 1 Exemplary compounds that can be synthesized using methods described in the present disclosure. Example of Biological Name Structure importance (Z)-3-hexanol

See, Sugimoto et al. (2014) (Z)-3-nonen-1-ol

West Indian Fruity Fly male sex pheromone (Z)-5-decen-1-ol

(Z)-5-decenyl acetate

Agrotis segetum sex pheromone component (E)-5-decen-1-ol

Anarsia lineatella sex pheromone component (E)-5-decenyl acetate

Anarsia lineatella sex pheromone component (Z)-7-dodecen-1-ol

(Z)-7-dodecenyl acetate

Pseudoplusia includens sex pheromone Agrotis segetum sex pheromone component (E)-8-dodecen-1-ol

Citrus Fruit Moth sex pheromone (E)-8-dodecenyl acetate

Grapholitha molesta, Ecdytolopha aurantiana sex pheromone component (Z)-8-dodecen-1-ol

Grapholitha molesta, Ecdytolopha aurantiana sex pheromone component (Z)-8-dodecenyl acetate

Grapholitha molesta sex pheromone component (Z)-9-dodecen-1-ol

(Z)-9-dodecenyl acetate

Eupoecilia ambiguella sex pheromone (Z)-9-tetradecen-1-ol

(Z)-9-tetradecenyl acetate

Pandemis pyrusana, Naranga aenescens, Agrotis segetum sex pheromone component (Z)-11-tetraceden-1-ol

(Z)-11-tetracedenyl acetate

Pandemis pyrusana, Choristoneura roseceana sex pheromone component (E)-11-tetradecen-1-ol

(E)-11-tetradecenyl acetate

Choristoneura roseceana, Crocidolomia pavonana sex pheromone component (Z)-7-hexadecen-1-ol

(Z)-7-hexadecenal

Diatraea considerata sex pheromone component (Z)-9-hexadecen-1-ol

(Z)-9-hexadecenal

Helicoverpa zea, Helicoverpa armigera, Heliothis virescens sex pheromone component (Z)-9-hexadecenyl acetate

Naranga aenescens sex pheromone component (Z)-11-hexadecen-1-ol

(Z)-11-hexadecenal

Platyptila carduidactyla, Heliothis virescens sex pheromone Helicoverpa zea, Helicoverpa armigera, Plutella xylostella, Diatraea considerate, Diatraea grandiosella, Diatraea saccharalis, Acrolepiopsis assectella sex pheromone component (Z)-11-hexadecenyl acetate

Discestra trifolii sex pheromone Heliothis virescens, Plutella xylostella, Acrolepiopsis assectella, Crocidolomia pavonana, Naranga aenescens sex pheromone component (Z)-13-octadecen-1-ol

(Z)-13-octadecenal

Diatraea considerata, Diatraea grandiosella sex pheromone component Ac = —(CO)CH₃

Useful unsaturated fatty acids and related compounds can also be prepared using the olefinic alcohol products as intermediates. In such cases, the methods of the invention generally include oxidizing the olefinic alcohol product to form an olefinic acid product.

The synthetic strategies disclosed herein chiefly rely on the ability of hydroxylases to terminally hydroxylate hydrocarbon substrates such as linear alkenes. Linear alkenes and other hydrocarbon substrates can be synthesized via any route, including but not limited to olefin metathesis, Wittig olefination, or alkyne substitution followed by partial hydrogenation. The hydroxylation products can further be modified via any method, including but not limited to oxidation, esterification, and olefin metathesis, to produce the desired end products (Scheme 2). Deviations from this general scheme are also disclosed.

Synthesis of Terminal Alkenols Via Metathesis and Hydroxylation

In a first aspect, the invention provides a method for synthesizing an olefinic alcohol product that includes incubating an unsaturated hydrocarbon substrate with an enzyme capable of selectively hydroxylating one terminal carbon of the unsaturated hydrocarbon substrate to form an unsaturated hydrocarbon alcohol. In some embodiments, the unsaturated hydrocarbon alcohol is the olefinic alcohol product. In some embodiments, the method further includes converting the unsaturated hydrocarbon alcohol to the olefinic alcohol product. In some embodiments, the unsaturated hydrocarbon substrate is an olefinic substrate. In some embodiments, the olefinic substrate is a metathesis product.

Hydroxylation of Symmetric Alkenes

In some embodiments, the method for synthesizing an oxyfunctionalized alkene includes a combination of metathesis and terminal hydroxylation as shown in Scheme 3.

According to Scheme 3, an E-selective metathesis catalyst or a Z-selective metathesis catalyst is used to convert a linear, terminal alkene of chain length m to the respective linear symmetric alkene. Following the metathesis step, a terminal hydroxylation biocatalyst is used to convert the symmetric alkene to the terminal alkenol. Optionally, this scheme can be expanded by an additional metathesis step that replaces the non-hydroxylated end of the terminal alkenol with one of another chain length (Scheme 4a).

In some instances, Scheme 4a can be further modified to protect the alcohol prior to metathesis. For example, the alcohol can be esterified prior to the metathesis step (Scheme 4b). The esterification is typically performed with formate or acetate, resulting in R═H or CH₃, respectively. The ester intermediates or final products can be hydrolysed to yield alcohol products.

The methods can be conducted with alkenes of any suitable length, which will depend on factors such as the desired olefinic alcohol product and the enzyme used in the biohydroxylation step. In some embodiments, the olefinic alcohol product is a C4-C20 olefinic alcohol product. The olefinic alcohol can contain, for example, 4-20 carbon atoms, or 8-20 carbon atoms, or 12-20 carbon atoms, or 16-20 carbon atoms. In such embodiments, the sum of the subscripts m, n, and y shown in Scheme 3 and Scheme 4 will bring the total number of carbon atoms in a particular olefinic alcohol product to 4-20, when added to the number of the non-subscripted carbon atoms shown in the structure for the olefinic alcohol product. In such embodiments, for example, subscript m in Scheme 3 can be an integer from 0-8, bringing the total number of the carbons in the symmetric olefinic substrate to 4-20. When m is 3, the route depicted in Scheme 3 provides (E/Z)-5-decen-1-ol.

Accordingly, some embodiments of the invention provide methods for preparing an olefinic alcohol product as described above, wherein the olefinic substrate is a metathesis product, and wherein the method includes: a) self-metathesizing a terminal olefin in the presence of a metathesis catalyst to form the metathesis product; and b) incubating the metathesis product with an enzyme capable of selectively hydroxylating one terminal carbon of the metathesis product to form an olefinic alcohol product.

In some embodiments, the terminal olefin has the formula (CH₂═CH)(CH₂)_(m)H, the metathesis product has the formula H(CH₂)_(m)(CH═CH)(CH₂)_(m)H, the olefinic alcohol product has the formula H(CH₂)_(m)(CH═CH)(CH₂)_(m)OH, and m is selected from an integer between 1 and 17. In some embodiments, m is selected from an integer between 1 and 9. In some embodiments, for example, m is 1, 2, 3, 4, 5, 6, 7, or 8. It is to be understood that any range disclosed in the present specification and recited in the claims includes the endpoints the endpoints of the range, unless explicitly stated otherwise. As a non-limiting example, integers in the range “between 1 and 17” include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17.

Hydroxylation of Asymmetric Alkenes

In some embodiments, the method for synthesizing an oxyfunctionalized alkene includes a combination of metathesis and terminal hydroxylation as shown in Scheme 5. In this process, terminal alkenes of different lengths are combined to generate asymmetric alkenes, which are then subjected to biohydroxylation conditions to afford the desired alkenol products.

Methods including hydroxylation of asymmetric alkenes can be conducted with alkenes of any suitable length. In some embodiments, the asymmetric olefinic alcohol product is a C4-C30 olefinic alcohol product. In such embodiments, the sum of the subscripts m and n shown in Scheme 5 will bring the total number of carbon atoms in a particular asymmetric olefinic alcohol product to 4-30, when added to the number of the non-subscripted carbon atoms shown in the structure for the asymmetric olefinic alcohol product. In such embodiments, for example, subscript m in Scheme 5 can be an integer from 8-18 and subscript n in Scheme 5 can be a different integer from 0-8, bringing the total number of the carbons in the asymmetric olefinic substrate to 4-30. When m is 9 and n is 3, the route depicted in Scheme 5 provides (E/Z)-hexadec-11-en-1-ol as the target product. In some embodiments, the asymmetric olefinic alcohol product is a C4-C20 olefinic alcohol product. The asymmetric olefinic alcohol can contain, for example, 4-20 carbon atoms, or 8-20 carbon atoms, or 12-20 carbon atoms, or 16-20 carbon atoms.

In some embodiments, for example, m is 0 and n is 4; or m is 1 and n is 3; or m is 3 and n is 1; or m is 4 and n is 0; or m is 0 and n is 5; or m is 1 and n is 4; or m is 2 and n is 3; or m is 3 and n is 2; or m is 4 and n is 1; or m is 5 and n is 0; or m is 0 and n is 6; or m is 1 and n is 5; or m is 2 and n is 4; or m is 4 and n is 2; or m is 5 and n is 1; or m is 6 and n is 0; or m is 0 and n is 7; or m is 1 and n is 6; or m is 2 and n is 5; or m is 3 and n is 4; or m is 4 and n is 3; or m is 5 and n is 2; or m is 6 and n is 1; or m is 7 and n is 0; or m is 0 and n is 8; or m is 1 and n is 7; or m is 2 and n is 6; or m is 3 and n is 5; or m is 5 and n is 3; or m is 6 and n is 2; or m is 7 and n is 1; or m is 8 and n is 0; or m is 0 and n is 9; or m is 1 and n is 8; or m is 2 and n is 7; or m is 3 and n is 6; or m is 4 and n is 5; or m is 5 and n is 4; or m is 6 and n is 3; or m is 7 and n is 2; or m is 8 and n is 1; or m is 9 and n is 0; or m is 0 and n is 10; or m is 1 and n is 9; or m is 2 and n is 8; or m is 3 and n is 7; or m is 4 and n is 6; or m is 6 and n is 4; or m is 7 and n is 3; or m is 8 and n is 2; or m is 9 and n is 1; or m is 10 and n is 0; or m is 0 and n is 11; or m is 1 and n is 10; or m is 2 and n is 9; or m is 3 and n is 8; or m is 4 and n is 7; or m is 5 and n is 6; or m is 6 and n is 5; or m is 7 and n is 4; or m is 8 and n is 3; or m is 9 and n is 2; or m is 10 and n is 1; or m is 11 and n is 0; or m is 0 and n is 12; or m is 1 and n is 11; or m is 2 and n is 10; or m is 3 and n is 9; or m is 4 and n is 8; or m is 5 and n is 7; or m is 7 and n is 5; or m is 8 and n is 4; or m is 9 and n is 3; or m is 10 and n is 2; or m is 11 and n is 1; or m is 12 and n is 0; or m is 0 and n is 13; or m is 1 and n is 12; or m is 2 and n is 11; or m is 3 and n is 10; or m is 4 and n is 9; or m is 5 and n is 8; or m is 6 and n is 7; or m is 7 and n is 6; or m is 8 and n is 5; or m is 9 and n is 4; or m is 10 and n is 3; or m is 11 and n is 2; or m is 12 and n is 1; or m is 13 and n is 0; or m is 0 and n is 14; or m is 1 and n is 13; or m is 2 and n is 12; or m is 3 and n is 11; or m is 4 and n is 10; or m is 5 and n is 9; or m is 6 and n is 8; or m is 8 and n is 6; or m is 9 and n is 5; or m is 10 and n is 4; or m is 11 and n is 3; or m is 12 and n is 2; or m is 13 and n is 1; or m is 14 and n is 0; or m is 0 and n is 15; or m is 1 and n is 14; or m is 2 and n is 13; or m is 3 and n is 12; or m is 4 and n is 11; or m is 5 and n is 10; or m is 6 and n is 9; or m is 7 and n is 8; or m is 8 and n is 7; or m is 9 and n is 6; or m is 10 and n is 5; or m is 11 and n is 4; or m is 12 and n is 3; or m is 13 and n is 2; or m is 14 and n is 1; or m is 15 and n is 0; or m is 0 and n is 16; or m is 1 and n is 15; or m is 2 and n is 14; or m is 3 and n is 13; or m is 4 and n is 12; or m is 5 and n is 11; or m is 6 and n is 10; or m is 7 and n is 9; or m is 9 and n is 7; or m is 10 and n is 6; or m is 11 and n is 5; or m is 12 and n is 4; or m is 13 and n is 3; or m is 14 and n is 2; or m is 15 and n is 1; or m is 16 and n is 0; or m is 1 and n is 16; or m is 2 and n is 15; or m is 3 and n is 14; or m is 4 and n is 13; or m is 5 and n is 12; or m is 6 and n is 11; or m is 7 and n is 10; or m is 8 and n is 9; or m is 9 and n is 8; or m is 10 and n is 7; or m is 11 and n is 6; or m is 12 and n is 5; or m is 13 and n is 4; or m is 14 and n is 3; or m is 15 and n is 2; or m is 16 and n is 1; or m is 17 and n is 0; or m is 0 and n is 17; or m is 1 and n is 17; or m is 2 and n is 16; or m is 3 and n is 15; or m is 4 and n is 14; or m is 5 and n is 13; or m is 6 and n is 12; or m is 7 and n is 11; or m is 8 and n is 10; or m is 10 and n is 8; or m is 11 and n is 7; or m is 12 and n is 6; or m is 13 and n is 5; or m is 14 and n is 4; or m is 15 and n is 3; or m is 16 and n is 2; or m is 17 and n is 1; or m is 18 and n is 0.

Accordingly, some embodiments of the invention provide methods for preparing an olefinic alcohol product as described above, wherein the olefinic substrate is a metathesis product, and wherein the method includes: a) cross-metathesizing a first terminal olefin and a second different terminal olefin in the presence of a metathesis catalyst to form the metathesis product; and b) incubating the metathesis product with an enzyme capable of selectively hydroxylating one terminal carbon of the metathesis product to form an olefinic alcohol product.

In some embodiments, the first terminal olefin has the formula (CH₂═CH)(CH₂)_(m)H, the second different terminal olefin has the formula (CH₂═CH)(CH₂)_(n)H, the metathesis product has the formula H(CH₂)_(m)(CH═CH)(CH₂)_(n)H, the olefinic alcohol product has the formula H(CH₂)_(m)(CH═CH)(CH₂)_(n)OH, and m and n are different integers between 1 and 17. In some embodiments, m and n are different integers between 1 and 9.

The methods of the invention can also be conducted such that the biohydroxylation step is conducted prior to the metathesis step and/or other synthetic transformation steps. Accordingly, some embodiments of the invention provide methods wherein the olefinic substrate is a first terminal olefin, and wherein the method includes: a) incubating the first terminal olefin with an enzyme capable of selectively hydroxylating the terminal carbon of the terminal olefin to form an α,ω-alkenol; and b) metathesizing the α,ω-alkenol and a second terminal olefin in the presence of a metathesis catalyst to form the olefinic alcohol product.

The alcohol can be protected with a suitable protecting group if necessary. In some embodiments, the methods of the invention include: a) incubating the first terminal olefin with an enzyme capable of selectively hydroxylating the terminal carbon of the terminal olefin to form an α,ω-alkenol; b) protecting the α,ω-alkenol to form a protected α,ω-alkenol; c) metathesizing the protected α,ω-alkenol and a second terminal olefin in the presence of a metathesis catalyst to form a protected olefinic alcohol product; and d) deprotecting the protected olefinic alcohol product to form the olefinic alcohol product.

Any suitable alcohol protecting group can be used in the methods of the invention. Such protecting groups are well known to one of ordinary skill in the art, including those that are disclosed in Protective Groups in Organic Synthesis, 4th edition, T. W. Greene and P. G. M. Wuts, John Wiley & Sons, New York, 2006, which is incorporated herein by reference in its entirety. In some embodiments, the α,ω-alkenol is protected via esterification and the protected olefinic alcohol product is deprotected via hydrolysis. In some embodiments, the α,ω-alkenol is protected via esterification with an acid selected from the group consisting of formate and acetate.

Any suitable olefinic substrate can be used in methods where the biohydroxylation step is conducted prior to the metathesis step and/or other synthetic transformation steps. In some embodiments, the first terminal olefin has the formula (CH₂═CH)(CH₂)_(m)H, the α,ω-alkenol has the formula (CH₂═CH)(CH₂)_(m)OH, the second terminal olefin has the formula (CH₂═CH)(CH₂)_(n)H, the olefinic alcohol product has the formula H(CH₂)_(n)(CH═CH)(CH₂)_(m)OH, and m and n are each independently selected from an integer between 1 and 17. In some embodiments, m and n are each independently selected from an integer between 1 and 9.

Hydroxylation of Alkanes to Terminal Diols followed by Monodehydration and Metathesis

Saturated hydrocarbon substrates can also be used in the methods of the invention. Accordingly, another aspect of the invention provides a method for synthesizing an olefinic alcohol product that includes: incubating a saturated hydrocarbon substrate with an enzyme capable of selectively hydroxylating one terminal carbon of the saturated hydrocarbon substrate to form a saturated hydrocarbon alcohol; and converting the saturated hydrocarbon alcohol to the olefinic alcohol product.

In some embodiments, terminal alkenols are synthesized according to Scheme 6a.

In this synthesis scheme, the terminal hydroxylation occurs on both ends of a linear alkane, resulting in a diol. The diol is then selectively monodehydrated through various chemical processes, including but not limited to those described in the literature (Sato et al., 2013) to generate a terminal alkenol. Coupling of the terminal alkenol with other alkenes via an olefin metathesis process allows for the synthesis of various pheromones.

In some instances, Scheme 6a can be further modified to protect the alcohol prior to metathesis. For example, the alcohol can be esterified prior to the metathesis step (Scheme 6b). The esterification is typically performed with formate or acetate, resulting in R═H or CH₃, respectively. The ester intermediates or final products can be hydrolysed to yield the alcohol products.

Accordingly, some embodiments of the invention provide a method wherein the saturated hydrocarbon substrate is an alkane substrate, and wherein the method includes:

-   -   a) incubating an alkane substrate with an enzyme capable of         selectively hydroxylating both terminal carbons of the alkane         substrate to form a terminal diol;     -   b) selectively dehydrating one of the terminal hydroxyl groups         of the terminal diol to form an α,ω-alkenol; and     -   c) metathesizing the α,ω-alkenol and a terminal olefin in the         presence of a metathesis catalyst to form the olefinic alcohol         product.

Accordingly, some embodiments of the invention provide a method wherein the saturated hydrocarbon substrate is an alkane substrate, and wherein the method includes:

-   -   a) incubating an alkane substrate with an enzyme capable of         selectively hydroxylating both terminal carbons of the alkane         substrate to form a terminal diol;     -   b) selectively dehydrating one of the terminal hydroxyl groups         of the terminal diol to form an α,ω-alkenol;     -   c) protecting the α,ω-alkenol to form a protected α,ω-alkenol;     -   d) metathesizing the protected α,ω-alkenol and a terminal olefin         in the presence of a metathesis catalyst to form a protected         olefinic alcohol product; and     -   e) deprotecting the protected olefinic alcohol product to form         the olefinic alcohol product.

In some embodiments, the α,ω-alkenol is protected via esterification and the protected olefinic alcohol product is deprotected via hydrolysis. In some embodiments, the α,ω-alkenol is protected via esterification with an acid selected from the group consisting of formate and acetate.

In some embodiments, the alkane substrate has the formula H(CH₂)_(m)H, the terminal diol has the formula HO(CH₂)_(m)OH, the α,ω-alkenol has the formula (CH₂═CH)(CH₂)_(m-2)OH, the terminal olefin has the formula (CH₂═CH) (CH₂)_(n)H, the olefinic alcohol product has the formula H(CH₂)_(n)(CH═CH)(CH₂)_(m-2)OH, m is an integer between 3 and 17, and n is an integer between 1 and 17. The alkane substrate, the terminal diol, the α,ω-alkenol, the terminal olefin, and the olefinic alcohol product can have any suitable combination of subscripts m and n, as described above. In some embodiments, m and n are independently selected integers between 1 and 9. In some embodiments, m and n are different integers between 1 and 9.

Hydroxylation of Terminal Alkenes

In some embodiments, terminal alkenols are synthesized according to Scheme 7a.

In a particular embodiment, the hydroxylase enzyme favors hydroxylation of the terminal CH₃ group over epoxidation of the C═C double bond.

In some instances, Scheme 7a can be further modified to protect the alcohol prior to metathesis. For example, the alcohol can be esterified prior to the metathesis step (Scheme 7b). The esterification is typically performed with formate or acetate, resulting in R═H or CH₃, respectively. The ester intermediates or final products can be hydrolysed to yield alcohol products.

The alkenes and the olefinic alcohol product can have any suitable combination of subscripts m and n, as described above. In some embodiments, m and n are independently selected integers between 1 and 9. In some embodiments, m and n are different integers between 1 and 9.

Hydroxylation of Fatty Acids

In some embodiments, terminal alkenols are synthesized according to Scheme 8a. In this Scheme, a terminal olefin forming fatty acid decarboxylase is used to convert a ω-hydroxy fatty acid into a fatty alkene.

In some instances, Scheme 8a can be further modified to protect the alcohol prior to metathesis. For example, the alcohol can be esterified prior to the metathesis step (Scheme 8b). The esterification is typically performed with formate or acetate, resulting in R═H or CH₃, respectively. The ester intermediates or final products can be hydrolysed to yield alcohol products.

The fatty acid substrate, the terminal hydroxy fatty acid, the alkenes, and the olefinic alcohol product can have any suitable combination of subscripts m and n, as described above. In some embodiments, m and n are independently selected integers between 1 and 9. In some embodiments, m and n are different integers between 1 and 9.

Alternative Synthesis of Terminal Alkenols Via Hydroxylation Hydroxylation of Symmetric or Asymmetric Alkenes

In some embodiments, symmetric or asymmetric alkenes are hydroxylated according to Schemes 3 and 4, respectively to produce symmetric or asymmetric alkenols. However, in this embodiment, the alkene is produced according to Scheme 9 (see, Oprean et al. (2006) for the acetylation step and Buck and Chong (2001) for the alkyne alkylation step), Scheme 10 (see, Buck and Chong (2001) regarding the alkyne alkylation step), Scheme 11a, or Scheme 11b. Scheme 11b shows Wittig reaction conditions that favor the formation of the Z-isomer according to Smith et al. (2000).

Accordingly, some embodiments of the invention provide a method for synthesizing an olefinic alcohol product wherein the method includes:

a) forming a reaction mixture comprising a terminal alkyne according to formula I

-   -   wherein n is an integer from 0 to 16,     -   and an alkyl halide according to formula II

-   -   wherein X is a halogen and m is an integer from 0 to 16,     -   under conditions sufficient to form a disubstituted alkyne         according to formula III

b) reducing the disubstituted alkyne to form an olefin according to formula IVa or IVb

-   -   and         c) incubating the olefin with an enzyme capable of selectively         hydroxylating one terminal carbon of the olefin to form the         olefinic alcohol product.

The terminal alkyne, the alkyl halide, the disubstituted alkyne, the olefin, and the olefinic alcohol product can have any suitable combination of subscripts m and n, as described above. In some embodiments, m and n are independently selected integers between 1 and 9. In some embodiments, m and n are different integers between 1 and 9.

In some embodiments, the invention includes:

a) forming a reaction mixture comprising a phosphonium salt according to formula XVI

-   -   wherein         -   each R is independently selected from C₁₋₆ alkyl and C₆₋₁₀             aryl,         -   X is a halogen, and         -   n is an integer from 0 to 16,     -   and an aldehyde according to formula XVII

-   -   wherein m is an integer from 0 to 16,     -   under conditions sufficient to form an olefin according to         formula XVIIIa or formula XVIIIb

-   -   and         b) incubating the olefin with an enzyme capable of selectively         hydroxylating one terminal carbon of the olefin to form the         olefinic alcohol product.

The phosphonium salt, the aldehyde, the olefin, and the olefinic alcohol product can have any suitable combination of subscripts m and n, as described above. In some embodiments, m and n are independently selected integers between 1 and 9. In some embodiments, m and n are different integers between 1 and 9.

Enzymatic or Other Oxidation of Diol to Aldehyde Followed by Wittig

In some embodiments, terminal alkenols are synthesized according to Scheme 12a. In certain embodiments, the Wittig reaction favors formation of the Z-olefin (Scheme 12b; see, Smith et al. 2000).

Accordingly, some embodiments of the invention provide methods that include:

-   -   a) incubating an alkane according to formula XIX

-   -   wherein m is an integer from 0 to 16,     -   with an enzyme capable of hydroxylating the terminal carbons of         the alkane to form a diol according to formula XX

-   -   b) oxidizing the diol to form an aldehyde according to formula         XXI

-   -   and     -   c) forming a reaction mixture comprising the aldehyde and a         phosphonium salt according to formula XXII

-   -   wherein         -   each R is independently selected from C₁₋₆ alkyl and C₆₋₁₀             aryl,         -   X is a halogen, and         -   n is an integer from 0 to 16,     -   under conditions sufficient to form an olefinic alcohol product         according to formula XXIIIa or formula XXIIIb

The alkane, the diol, the aldehyde, the phosphonium salt, and the olefinic alcohol product can have any suitable combination of subscripts m and n, as described above. In some embodiments, m and n are independently selected integers between 1 and 9. In some embodiments, m and n are different integers between 1 and 9.

Hydroxylation of Terminal Alkynes

In some embodiments, terminal alkenols are synthesized by first hydroxylating terminal alkynes to their corresponding alkynols. Upon protection of the hydroxyl functional group, the alkynols can be further functionalized by alkylation with other haloalkanes to generate disubstituted alkynols and partial hydrogenation to afford the desired alkene products, as shown in Scheme 13.

Accordingly, some embodiments of the invention provide a method wherein the hydrocarbon substrate is an alkyne and the method includes:

a) incubating a terminal alkyne according to formula V

-   -   wherein m is an integer from 0 to 16,     -   with an enzyme capable of selectively hydroxylating one terminal         carbon of the terminal alkyne to form an unsaturated hydrocarbon         alcohol according to formula VI

b) forming a reaction mixture comprising the unsaturated hydrocarbon alcohol and an alkyl halide according to formula VII

-   -   wherein X is a halogen and n is an integer from 0 to 16,     -   under conditions sufficient to form a disubstituted alkyne         according to formula VIII

-   -   and         c) reducing the disubstituted alkyne to form an olefinic alcohol         product according to formula IXa or IXb

In some embodiments, the unsaturated hydrocarbon alcohol according to formula VI is protected, and then the resulting protected unsaturated hydrocarbon alcohol is combined with an alkyl halide according to formula VII under conditions sufficient to form a protected disubstituted alkyne. The protected disubstituted alkyne can be reduced and deprotected to provide an olefinic alcohol product according to formula IXa or IXb.

The terminal alkyne, the unsaturated hydrocarbon alcohol, the alkyl halide, the disubstituted alkyne, and the olefinic alcohol product can have any suitable combination of subscripts m and n, as described above. In some embodiments, m and n are independently selected integers between 1 and 9. In some embodiments, m and n are different integers between 1 and 9.

Hydroxylation of Halogenated Alkanes

In some embodiments, terminal halogenated alkanes can be biohydroxylated to generate α,ω-halogenated alcohols. Upon protection of the alcohol moiety, the substrate can be coupled with a terminal alkyne to afford an internal alkyne product, which can be partially reduced via known chemical processes to generate either the cis- or trans-alkenes that can be readily converted to insect pheromones, as illustrated in Scheme 14.

Accordingly, some embodiments of the invention provide a method wherein the saturated hydrocarbon substrate is an alkyl halide, and wherein the method includes:

a) incubating an alkyl halide according to formula X

-   -   wherein X is a halogen and n is an integer from 0 to 16,     -   with an enzyme capable of selectively hydroxylating one terminal         carbon of the alkyl halide to form a halogen-substituted alkanol         according to formula XI

b) converting the halogen-substituted alkanol to a protected halogen-substituted alkanol according to formula XII

-   -   wherein R is a protecting group;         c) forming a reaction mixture comprising the protected         halogen-substituted alkanol and a terminal alkyne according to         formula XIII

-   -   wherein m is an integer from 0 to 16,     -   under conditions sufficient to form a disubstituted alkyne         according to formula XIV

d) reducing the disubstituted alkyne to form a protected olefinic alcohol according to formula XVa or formula XVb

-   -   and         e) converting the protected olefinic alcohol to the olefinic         alcohol product.

The alkyl halide, the halogen-substituted alkanol, the protected halogen-substituted alkanol, the terminal alkyne, the disubstituted alkyne, and the olefinic alcohol product can have any suitable combination of subscripts m and n, as described above. In some embodiments, m and n are independently selected integers between 1 and 9. In some embodiments, m and n are different integers between 1 and 9.

Hydroxylation of Halogenated Alkenes

In some embodiments, halogenated alkenes can be biohydroxylated to generate corresponding alkenols. Upon protection of the alcohol moiety, the substrate is then coupled with haloalkanes, alkenes, or alkynes to provide suitable intermediates for synthesis of insect pheromones as illustrated in Scheme 15 below.

Accordingly, some embodiments of the invention provide a method wherein the unsaturated hydrocarbon substrate is an alkenyl halide, and wherein the method includes:

a) incubating an alkenyl halide according to formula X′

-   -   wherein X is a halogen,     -   a and c are independently integers from 1 to 15, and     -   b is an integer from 1 to 3,     -   with an enzyme capable of selectively hydroxylating one terminal         carbon of the alkenyl halide to form a halogen-substituted         unsaturated hydrocarbon alcohol according to formula XI′

b) converting the halogen-substituted unsaturated hydrocarbon alcohol to a protected halogen-substituted unsaturated hydrocarbon alcohol according to formula XII′

-   -   wherein R is a protecting group;         c) forming a reaction mixture comprising the protected         halogen-substituted unsaturated hydrocarbon alcohol and a         reactant according to formula XIIa′

-   -   wherein Y is a halogen and     -   d is an interger from 0 to 15     -   under conditions sufficient to form an elongated protected         unsaturated hydrocarbon alcohol according to formula XIIb′

-   -   and         d) converting the elongated protected unsaturated hydrocarbon         alcohol to the olefinic alcohol product.

Dehydration of Diols to Alkyne Followed by Alkyne Substitution and Reduction

In some embodiments, terminal alkenols are synthesized according to Scheme 16.

Accordingly, some embodiments of the invention provide a method which includes:

a) incubating an alkane according to formula XXIX

-   -   wherein m is an integer from 0 to 16,     -   with an enzyme capable of hydroxylating the terminal carbons of         the alkane to form a diol according to formula XXX

b) converting the diol to an alkyne according to formula XXXI

c) forming a reaction mixture comprising the alkyne and an alkyl halide according to formula XXXII

-   -   wherein     -   X is a halogen and n is an integer from 0 to 15,     -   under conditions sufficient to form a disubstituted alkyne         according to formula XXXIII

-   -   and         d) reducing the disubstituted alkyne to form an olefinic alcohol         product according to formula XXXIVa or formula XXXIVb

The alkane, the diol, the alkyne, the alkyl halide, the disubstituted alkyne, and the olefinic alcohol product can have any suitable combination of subscripts m and n, as described above. In some embodiments, m and n are independently selected integers between 1 and 9. In some embodiments, m and n are different integers between 1 and 9.

Hydroxylation of Fatty Acids Followed by a Reduction and Wittig Olefination

In some embodiments, terminal alkenols are synthesized according to Scheme 17a. Under certain conditions, the Wittig reaction favors the formation of the Z-isomer (Scheme 17b; see, Smith et al. 2000).

Accordingly, some embodiments of the invention provide a method wherein the saturated hydrocarbon substrate is a fatty acid, and wherein the method includes:

-   -   a) incubating a fatty acid with an enzyme capable of selectively         hydroxylating the terminal carbon of the fatty acid to form a         terminal hydroxy fatty acid;     -   b) selectively decarbonylating the carboxyl group of the         terminal hydroxy fatty acid to form an α,ω-alkenol; and     -   c) metathesizing the α,ω-alkenol and a terminal olefin in the         presence of a metathesis catalyst to form the olefinic alcohol         product.

In some embodiments, the invention provides a method wherein the saturated hydrocarbon substrate is a fatty acid, and wherein the method includes:

-   -   a) incubating a fatty acid with an enzyme capable of selectively         hydroxylating the terminal carbon of the fatty acid to form a         terminal hydroxy fatty acid;     -   b) selectively decarbonylating the carboxyl group of the         terminal hydroxy fatty acid to form an α,ω-alkenol;     -   c) protecting the α,ω-alkenol to form a protected α,ω-alkenol;     -   d) metathesizing the protected α,ω-alkenol and a terminal olefin         in the presence of a metathesis catalyst to form a protected         olefinic alcohol product; and     -   e) deprotecting the protected olefinic alcohol product to form         the olefinic alcohol product.

In some embodiments, the α,ω-alkenol is protected via esterification and the protected olefinic alcohol product is deprotected via hydrolysis. In some embodiments, the α,ω-alkenol is protected via esterification with an acid selected from the group consisting of formate and acetate.

In some embodiments wherein the saturated hydrocarbon substrate is a fatty acid, the fatty acid has the formula H(CH₂)_(m)CO₂H, the terminal hydroxy fatty acid has the formula HO(CH₂)_(m)CO₂H, the α,ω-alkenol has the formula (CH₂═CH)(CH₂)_(m-2)OH, the terminal olefin has the formula (CH₂═CH)(CH₂)_(n)H, the olefinic alcohol product has the formula H(CH₂)_(n)(CH═CH)(CH₂)_(m-2)OH, and m and n are each independently selected from an integer between 3 and 17.

The fatty acid, the terminal hydroxy fatty acid, the α,ω-alkenol, the terminal olefin, and the olefinic alcohol product can have any suitable combination of subscripts m and n, as described above. In some embodiments, m and n are different integers between 1 and 9.

In some embodiments, the invention provides a method wherein the saturated hydrocarbon substrate is a fatty acid, and wherein the method includes:

a) incubating a fatty acid according to formula XXIV

-   -   wherein m is an integer from 0 to 14,     -   with an enzyme capable of hydroxylating the terminal carbon of         the fatty acid to form a terminal hydroxy fatty acid according         to formula XXV

b) reducing the terminal hydroxy fatty acid to form an aldehyde according to formula XXVI

-   -   and         c) forming a reaction mixture comprising the aldehyde and a         phosphonium salt according to formula XXVII

-   -   wherein         -   each R is independently selected from C₁₋₆ alkyl and C₆₋₁₀             aryl,         -   X is a halogen, and         -   n is an integer from 0 to 14,     -   under conditions sufficient to form an olefinic alcohol product         according to formula XXVIIIa or formula XXVIIIb

The alkane, fatty acid, the terminal hydroxy fatty acid, the aldehyde, the phosponium salt, and the olefinic alcohol product can have any suitable combination of subscripts m and n, as described above. In some embodiments, m and n are independently selected integers between 1 and 9. In some embodiments, m and n are different integers between 1 and 9.

Synthesis of Oxyfunctionalized Olefins Comprising More than One C═C Double Bond

Hydroxylation of Conjugated and Unconjugated Alkenes

In some embodiments, conjugated and unconjugated alkenes can be biohydroxylated to generate corresponding conjugated and unconjugated alkenols. The resulting products are then coupled with haloalkanes, haloalkenes, or alkynes to provide suitable intermediates for synthesis of insect pheromones as illustrated in Scheme 18 below.

Accordingly, some embodiments of the invention provide a method wherein the unsaturated hydrocarbon substrate is an alkenyl halide, and wherein the method includes:

a) incubating an alkenyl halide according to formula X″

-   -   wherein X a halogen,     -   a, c, and e are independently integers from 0 to 15, and     -   b and d are independently integers from 0 to 2, provided that at         least one of b and d is other than 0,     -   with an enzyme capable of selectively hydroxylating one terminal         carbon of the alkene to form a halogen-substituted unsaturated         hydrocarbon alcohol according to formula XI″

b) converting the halogen-substituted unsaturated hydrocarbon alcohol to a protected halogen-substituted unsaturated hydrocarbon alcohol according to formula XII″

-   -   wherein R is a protecting group;         c) forming a reaction mixture comprising the protected         halogen-substituted unsaturated hydrocarbon alcohol and an alkyl         halide according to formula XIIa″

-   -   wherein Y is a halogen and     -   f is an integer from 0 to 15     -   under conditions sufficient to form an elongated protected         unsaturated hydrocarbon alcohol according to formula XIIb″

-   -   and         d) converting the elongated protected unsaturated hydrocarbon         alcohol to the olefinic alcohol product.

The alkenyl halide, the halogen-substituted unsaturated hydrocarbon alcohol, the protected halogen-substituted unsaturated hydrocarbon alcohol, the alkyl halide according to formula XIIa″, the elongated protected unsaturated hydrocarbon alcohol, and the olefinic alcohol product can have any suitable combination of subscripts a, b, c, d, e, and f. In some embodiments, a, b, c, d, e, and f are integers independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9. In some embodiments, the sum of a, b, c, d, e, and f is an integer from 1 to 14. In some embodiments, the sum of a, b, c, d, e, and f is an integer from 1 to 8.

Hydroxylation of Conjugated and Unconjugated Alkene-Alkynes

In some embodiments, conjugated and unconjugated alkene-alkyne bi-functional substrates can be biohydroxylated to generate alkene-alkynols. The resulting biohydroxylation products can then be used for coupling with haloalkanes, alkenes, or alkynes to provide suitable intermediates for synthesis of insect pheromones as illustrated in Scheme 19 below.

Accordingly, some embodiments of the invention provide a method which includes:

a) incubating an unsaturated halogen-substituted hydrocarbon according to formula X″′

-   -   wherein X is a halogen,     -   a, c, and e are independently integers from 0 to 30, and     -   b and d are independently integers from 1 to 2, provided that at         least one of b and d is other than 0,     -   with an enzyme capable of selectively hydroxylating one terminal         carbon of the halogen-substituted unsaturated hydrocarbon to         form a halogen-substituted unsaturated hydrocarbon alcohol         according to formula XI′″

b) converting the halogen-substituted unsaturated hydrocarbon alcohol to a protected halogen-substituted unsaturated hydrocarbon alcohol according to formula XII′″

-   -   wherein R is a protecting group;         c) forming a reaction mixture comprising the protected         halogen-substituted unsaturated hydrocarbon alcohol and an alkyl         halide according to formula XIIa″′

-   -   wherein Y is a halogen and     -   f is an interger from 0 to 15     -   under conditions sufficient to form an elongated protected         unsaturated hydrocarbon alcohol according to formula XIIb′″

-   -   and         d) converting the elongated protected unsaturated hydrocarbon         alcohol to the olefinic alcohol product.

The unsaturated halogen-substituted hydrocarbon, the halogen-substituted unsaturated hydrocarbon alcohol, the protected halogen-substituted unsaturated hydrocarbon alcohol, the alkyl halide according to formula XIIa′″, the elongated protected unsaturated hydrocarbon alcohol, and the olefinic alcohol product can have any suitable combination of subscripts a, b, c, d, e, and f. In some embodiments, a, b, c, d, e, and f are integers independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9. In some embodiments, the sum of a, b, c, d, e, and f is an integer from 1 to 14. In some embodiments, the sum of a, b, c, d, e, and f is an integer from 1 to 8.

Metathesis Catalysts

In general, any metathesis catalyst stable under the reaction conditions and nonreactive with the functional groups present on the reactant shown in Schemes 3 through 8 may be used with the present invention. Such catalysts are, for example, those described by Grubbs (Grubbs, R. H., “Synthesis of large and small molecules using olefin metathesis catalysts.” PMSE Prepr., 2012), herein incorporated by reference in its entirety. Depending on the desired isomer of the olefin, as cis-selective metathesis catalyst may be used, for example one of those described by Shahane et al. (Shahane, S., et al. Chem Cat Chem, 2013. 5(12): p. 3436-3459), herein incorporated by reference in its entirety. Specific catalysts 1-5 exhibiting cis-selectivity are shown below (Scheme 19a) and have been described previously (Khan, R. K., et al. J. Am. Chem. Soc., 2013. 135(28): p. 10258-61; Hartung, J. et al. J. Am. Chem. Soc., 2013. 135(28): p. 10183-5.; Rosebrugh, L. E., et al. J. Am. Chem. Soc., 2013. 135(4): p. 1276-9.; Marx, V. M., et al. J. Am. Chem. Soc., 2013. 135(1): p. 94-7.; Herbert, M. B., et al. Angew. Chem. Int. Ed. Engl., 2013. 52(1): p. 310-4; Keitz, B. K., et al. J. Am. Chem. Soc., 2012. 134(4): p. 2040-3; Keitz, B. K., et al. J. Am. Chem. Soc., 2012. 134(1): p. 693-9; Endo, K. et al. J. Am. Chem. Soc., 2011. 133(22): p. 8525-7).

Additional Z-selective catalysts are described in (Cannon and Grubbs 2013; Bronner et al. 2014; Hartung et al. 2014; Pribisko et al. 2014; Quigley and Grubbs 2014) and are herein incorporated by reference in their entirety. Due to their excellent stability and functional group tolerance, preferred metathesis catalysts include, but are not limited to, neutral ruthenium or osmium metal carbene complexes that possess metal centers that are formally in the +2 oxidation state, have an electron count of 16, are penta-coordinated, and are of the general formula LL′AA′M=CRbRc or LL′AA′M=(C═)nCRbRc (Pederson and Grubbs 2002); wherein

-   -   M is ruthenium or osmium;     -   L and L′ are each independently any neutral electron donor         ligand and preferably selected from phosphine, sulfonated         phosphine, phosphite, phosphinite, phosphonite, arsine,         stibnite, ether, amine, amide, imine, sulfoxide, carboxyl,         nitrosyl, pyridine, thioether, or heterocyclic carbenes; and     -   A and A′ are anionic ligands independently selected from         halogen, hydrogen, C₁-C₂₀ alkyl, aryl, C₁-C₂₀ alkoxide,         aryloxide, C₂-C₂₀ alkoxycarbonyl, arylcarboxylate, C₁-C₂₀         carboxylate, arylsulfonyl, C₁-C₂₀ alkylsulfonyl, C₁-C₂₀         alkylsulfinyl; each ligand optionally being substituted with         C₁-C₅ alkyl, halogen, C₁-C₅ alkoxy; or with a phenyl group that         is optionally substituted with halogen, C₁-C₅ alkyl, or C₁-C₅         alkoxy; and A and A′ together may optionally comprise a         bidentate ligand; and     -   R_(b) and R_(c) are independently selected from hydrogen, C₁-C₂₀         alkyl, aryl, C₁-C₂₀ carboxylate, C₁-C₂₀ alkoxy, aryloxy, C₁-C₂₀         alkoxycarbonyl, C₁-C₂₀ alkylthio, C₁-C₂₀ alkylsulfonyl and         C₁-C₂₀ alkylsulfinyl, each of R_(b) and R_(c) optionally         substituted with C₁-C₅ alkyl, halogen, C₁-C₅ alkoxy or with a         phenyl group that is optionally substituted with halogen, C₁-C₅         alkyl, or C₁-C₅ alkoxy.

Other metathesis catalysts such as “well defined catalysts” can also be used. Such catalysts include, but are not limited to, Schrock's molybdenum metathesis catalyst, 2,6-diisopropylphenylimido neophylidenemolybdenum (VI) bis(hexafluoro-t-butoxide), described by Grubbs et al. (Tetrahedron 1998, 54: 4413-4450) and Basset's tungsten metathesis catalyst described by Couturier, J. L. et al. (Angew. Chem. Int. Ed. Engl. 1992, 31: 628).

Catalysts useful in the methods of the invention also include those described by Peryshkov, et al. J. Am. Chem. Soc. 2011, 133: 20754-20757; Wang, et al. Angewandte Chemie, 2013, 52: 1939-1943; Yu, et al. J. Am. Chem. Soc., 2012, 134: 2788-2799; Halford. Chem. Eng. News, 2011, 89 (45): 11; Yu, et al. Nature, 2011, 479: 88-93; Lee. Nature, 2011, 471: 452-453; Meek, et al. Nature, 2011: 471, 461-466; Flook, et al. J. Am. Chem. Soc. 2011, 133: 1784-1786; Zhao, et al. Org Lett., 2011, 13(4): 784-787; Ondi, et al. “High activity, stabilized formulations, efficient synthesis and industrial use of Mo- and W-based metathesis catalysts” XiMo Technology Updates, 2015: http://www.ximo-inc.com/files/ximo/uploads/download/Summary_3.11.15.pdf; Schrock, et al. Macromolecules, 2010: 43, 7515-7522; Peryshkov, et al. Organometallics 2013: 32, 5256-5259; Gerber, et al. Organometallics 2013: 32, 5573-5580; Marinescu, et al. Organometallics 2012: 31, 6336-6343; Wang, et al. Angew. Chem. Int. Ed. 2013: 52, 1939-1943; Wang, et al. Chem. Eur. J. 2013: 19, 2726-2740; and Townsend et al. J. Am. Chem. Soc. 2012: 134, 11334-11337.

Catalysts useful in the methods of the invention also include those described in International Pub. No. WO 2014/155185; International Pub. No. WO 2014/172534; U.S. Pat. Appl. Pub. No. 2014/0330018; International Pub. No. WO 2015/003815; and International Pub. No. WO 2015/003814.

Catalysts useful in the methods of the invention also include those described in U.S. Pat. No. 4,231,947; U.S. Pat. No. 4,245,131; U.S. Pat. No. 4,427,595; U.S. Pat. No. 4,681,956; U.S. Pat. No. 4,727,215; International Pub. No. WO 1991/009825; U.S. Pat. No. 5,087,710; U.S. Pat. No. 5,142,073; U.S. Pat. No. 5,146,033; International Pub. No. WO 1992/019631; U.S. Pat. No. 6,121,473; U.S. Pat. No. 6,346,652; U.S. Pat. No. 8,987,531; U.S. Pat. Appl. Pub. No. 2008/0119678; International Pub. No. WO 2008/066754; International Pub. No. WO 2009/094201; U.S. Pat. Appl. Pub. No. 2011/0015430; U.S. Pat. Appl. Pub. No. 2011/0065915; U.S. Pat. Appl. Pub. No. 2011/0077421; International Pub. No. WO 2011/040963; International Pub. No. WO 2011/097642; U.S. Pat. Appl. Pub. No. 2011/0237815; U.S. Pat. Appl. Pub. No. 2012/0302710; International Pub. No. WO 2012/167171; U.S. Pat. Appl. Pub. No. 2012/0323000; U.S. Pat. Appl. Pub. No. 2013/0116434; International Pub. No. WO 2013/070725; U.S. Pat. Appl. Pub. No. 2013/0274482; U.S. Pat. Appl. Pub. No. 2013/0281706; International Pub. No. WO 2014/139679; International Pub. No. WO 2014/169014; U.S. Pat. Appl. Pub. No. 2014/0330018; and U.S. Pat. Appl. Pub. No. 2014/0378637.

Catalysts useful in the methods of the invention also include those described in International Pub. No. WO 2007/075427; U.S. Pat. Appl. Pub. No. 2007/0282148; International Pub. No. WO 2009/126831; International Pub. No. WO 2011/069134; U.S. Pat. Appl. Pub. No. 2012/0123133; U.S. Pat. Appl. Pub. No. 2013/0261312; U.S. Pat. Appl. Pub. No. 2013/0296511; International Pub. No. WO 2014/134333; and U.S. Pat. Appl. Pub. No. 2015/0018557.

Catalysts useful in the methods of the invention also include those set forth in the following table:

Structure Name

dichloro[1,3-bis(2,6-isopropylphenyl)-2- imidazolidinylidene](benzylidene)(tricyclohexyl- phosphine)ruthenium(II)

dichloro[1,3-bis(2,6-isopropylphenyl)-2- imidazolidinylidene](2- isopropoxyphenylmethylene)ruthenium(II)

dichloro[1,3-Bis(2-methylphenyl)-2- imidazolidinylidene](benzylidene)(tricyclohexyl- phosphine)ruthenium(II)

dichloro[1,3-bis(2-methylphenyl)-2- imidazolidinylidene](2- isopropoxyphenylmethylene)ruthenium(II)

dichloro[1,3-bis(2,4,6-trimethylphenyl)-2- imidazolidinylidene](benzylidene)bis(3- bromopyridine)ruthenium(II)

dichloro[1,3-bis(2,4,6-trimethylphenyl)-2- imidazolidinylidene](3-methyl-2- butenylidene)(tricyclohexylphosphine) ruthenium(II)

dichloro[1,3-bis(2,4,6-trimethylphenyl)-2- imidazolidinylidene][3-(2-pyridinyl) propylidene]ruthenium(II)

dichloro[1,3-bis(2,4,6-trimethylphenyl)-2- imidazolidinylidene][(tricyclohexylphosphoranyl) methylidene]ruthenium(II) tetrafluoroborate

dichloro(3-methyl-2-butenylidene) bis(tricyclohexylphosphine)ruthenium(II)

dichloro(3-methyl-2-butenylidene) bis(tricyclopentylphosphine)ruthenium(II)

dichloro(tricyclohexylphosphine)[(tricyclohexyl- phosphoranyl)methylidene]ruthenium(II) tetrafluoroborate

bis(tricyclohexylphosphine) benzylidine ruthenium(IV) dichloride

[1,3-bis-(2,4,6-trimethylpheny)-2- imidazolidinylidene]dichloro(phenylmethylene) (tricyclohexylphosphine)ruthenium

(1,3-bis-(2,4,6-trimethylphenyl)-2- imidazolidinylidene)dichloro(o- isopropoxyphenylmethylene)ruthenium

dichloro(o- isopropoxyphenylmethylene)(tricyclohexyl- phosphine)ruthenium(II)

[2-(1-methylethoxy-O)phenylmethyl- C](nitrato-O,O′){rel-(2R,5R,7R)-adamantane- 2,1-diyl[3-(2,4,6-trimethylphenyl)-1- imidazolidinyl-2-y lidene]}ruthenium

Catalysts useful in the methods of the invention also include those described in U.S. Pat. Appl. Pub. No. 2008/0009598; U.S. Pat. Appl. Pub. No. 2008/0207911; U.S. Pat. Appl. Pub. No. 2008/0275247; U.S. Pat. Appl. Pub. No. 2011/0040099; U.S. Pat. Appl. Pub. No. 2011/0282068; and U.S. Pat. Appl. Pub. No. 2015/0038723.

Catalysts useful in the methods of the invention include those described in International Pub. No. WO 2007/140954; U.S. Pat. Appl. Pub. No. 2008/0221345; International Pub. No. WO 2010/037550; U.S. Pat. Appl. Pub. No. 2010/0087644; U.S. Pat. Appl. Pub. No. 2010/0113795; U.S. Pat. Appl. Pub. No. 2010/0174068; International Pub. No. WO 2011/091980; International Pub. No. WO 2012/168183; U.S. Pat. Appl. Pub. No. 2013/0079515; U.S. Pat. Appl. Pub. No. 2013/0144060; U.S. Pat. Appl. Pub. No. 2013/0211096; International Pub. No. WO 2013/135776; International Pub. No. WO 2014/001291; International Pub. No. WO 2014/067767; U.S. Pat. Appl. Pub. No. 2014/0171607; and U.S. Pat. Appl. Pub. No. 2015/0045558.

The catalyst is typically provided in the reaction mixture in a sub-stoichiometric amount (e.g., catalytic amount). In certain embodiments, that amount is in the range of about 0.001 to about 50 mol % with respect to the limiting reagent of the chemical reaction, depending upon which reagent is in stoichiometric excess. In some embodiments, the catalyst is present in less than or equal to about 40 mol % relative to the limiting reagent. In some embodiments, the catalyst is present in less than or equal to about 30 mol % relative to the limiting reagent. In some embodiments, the catalyst is present in less than about 20 mol %, less than about 10 mol %, less than about 5 mol %, less than about 2.5 mol %, less than about 1 mol %, less than about 0.5 mol %, less than about 0.1 mol %, less than about 0.015 mol %, less than about 0.01 mol %, less than about 0.0015 mol %, or less, relative to the limiting reagent. In some embodiments, the catalyst is present in the range of about 2.5 mol % to about 5 mol %, relative to the limiting reagent. In some embodiments, the reaction mixture contains about 0.5 mol % catalyst. In the case where the molecular formula of the catalyst complex includes more than one metal, the amount of the catalyst complex used in the reaction may be adjusted accordingly.

In some cases, the methods described herein can be performed in the absence of solvent (e.g., neat). In some cases, the methods can include the use of one or more solvents. Examples of solvents that may be suitable for use in the invention include, but are not limited to, benzene, p-cresol, toluene, xylene, diethyl ether, glycol, diethyl ether, petroleum ether, hexane, cyclohexane, pentane, methylene chloride, chloroform, carbon tetrachloride, dioxane, tetrahydrofuran (THF), dimethyl sulfoxide, dimethylformamide, hexamethyl-phosphoric triamide, ethyl acetate, pyridine, triethylamine, picoline, and the like, as well as mixtures thereof. In some embodiments, the solvent is selected from benzene, toluene, pentane, methylene chloride, and THF. In certain embodiments, the solvent is benzene.

In some embodiments, the method is performed under reduced pressure. This may be advantageous in cases where a volatile byproduct, such as ethylene, may be produced during the course of the metathesis reaction. For example, removal of the ethylene byproduct from the reaction vessel may advantageously shift the equilibrium of the metathesis reaction towards formation of the desired product. In some embodiments, the method is performed at a pressure of about less than 760 torr. In some embodiments, the method is performed at a pressure of about less than 700 torr. In some embodiments, the method is performed at a pressure of about less than 650 torr. In some embodiments, the method is performed at a pressure of about less than 600 torr. In some embodiments, the method is performed at a pressure of about less than 550 torr. In some embodiments, the method is performed at a pressure of about less than 500 torr. In some embodiments, the method is performed at a pressure of about less than 450 torr. In some embodiments, the method is performed at a pressure of about less than 400 torr. In some embodiments, the method is performed at a pressure of about less than 350 torr. In some embodiments, the method is performed at a pressure of about less than 300 torr. In some embodiments, the method is performed at a pressure of about less than 250 torr. In some embodiments, the method is performed at a pressure of about less than 200 torr. In some embodiments, the method is performed at a pressure of about less than 150 torr. In some embodiments, the method is performed at a pressure of about less than 100 torr. In some embodiments, the method is performed at a pressure of about less than 90 torr. In some embodiments, the method is performed at a pressure of about less than 80 torr. In some embodiments, the method is performed at a pressure of about less than 70 torr. In some embodiments, the method is performed at a pressure of about less than 60 torr. In some embodiments, the method is performed at a pressure of about less than 50 torr. In some embodiments, the method is performed at a pressure of about less than 40 torr. In some embodiments, the method is performed at a pressure of about less than 30 torr. In some embodiments, the method is performed at a pressure of about less than 20 torr. In some embodiments, the method is performed at a pressure of about 20 torr.

In some embodiments, the method is performed at a pressure of about 19 torr. In some embodiments, the method is performed at a pressure of about 18 torr. In some embodiments, the method is performed at a pressure of about 17 torr. In some embodiments, the method is performed at a pressure of about 16 torr. In some embodiments, the method is performed at a pressure of about 15 torr. In some embodiments, the method is performed at a pressure of about 14 torr. In some embodiments, the method is performed at a pressure of about 13 torr. In some embodiments, the method is performed at a pressure of about 12 torr. In some embodiments, the method is performed at a pressure of about 11 torr. In some embodiments, the method is performed at a pressure of about 10 torr. In some embodiments, the method is performed at a pressure of about 10 torr. In some embodiments, the method is performed at a pressure of about 9 torr. In some embodiments, the method is performed at a pressure of about 8 torr. In some embodiments, the method is performed at a pressure of about 7 torr. In some embodiments, the method is performed at a pressure of about 6 torr. In some embodiments, the method is performed at a pressure of about 5 torr. In some embodiments, the method is performed at a pressure of about 4 torr. In some embodiments, the method is performed at a pressure of about 3 torr. In some embodiments, the method is performed at a pressure of about 2 torr. In some embodiments, the method is performed at a pressure of about 1 torr. In some embodiments, the method is performed at a pressure of less than about 1 torr.

In some embodiments, the two metathesis reactants are present in equimolar amounts. In some embodiments, the two metathesis reactants are not present in equimolar amounts. In certain embodiments, the two reactants are present in a molar ratio of about 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20. In certain embodiments, the two reactants are present in a molar ratio of about 10:1. In certain embodiments, the two reactants are present in a molar ratio of about 7:1. In certain embodiments, the two reactants are present in a molar ratio of about 5:1. In certain embodiments, the two reactants are present in a molar ratio of about 2:1. In certain embodiments, the two reactants are present in a molar ratio of about 1:10. In certain embodiments, the two reactants are present in a molar ratio of about 1:7. In certain embodiments, the two reactants are present in a molar ratio of about 1:5. In certain embodiments, the two reactants are present in a molar ratio of about 1:2.

In general, the reactions with many of the metathesis catalysts disclosed herein provide yields better than 15%, preferably better than 50%, more preferably better than 75%, and most preferably better than 90%. In addition, the reactants and products are chosen to provide at least a 5° C. difference, preferably a greater than 20° C. difference, and most preferably a greater than 40° C. difference in boiling points. Additionally, the use of metathesis catalysts allows for much faster product formation than byproduct, it is desirable to run these reactions as quickly as practical. In particular, the reactions are performed in less than about 24 hours, preferably less than 12 hours, more preferably less than 8 hours, and most preferably less than 4 hours.

One of skill in the art will appreciate that the time, temperature and solvent can depend on each other, and that changing one can require changing the others to prepare the pyrethroid products and intermediates in the methods of the invention. The metathesis steps can proceed at a variety of temperatures and times. In general, reactions in the methods of the invention are conducted using reaction times of several minutes to several days. For example, reaction times of from about 12 hours to about 7 days can be used. In some embodiments, reaction times of 1-5 days can be used. In some embodiments, reaction times of from about 10 minutes to about 10 hours can be used. In general, reactions in the methods of the invention are conducted at a temperature of from about 0° C. to about 200° C. For example, reactions can be conducted at 15-100° C. In some embodiments, reaction can be conducted at 20-80° C. In some embodiments, reactions can be conducted at 100-150° C.

Hydroxylation Catalysts

Various enzymes and/or whole cells comprising enzymes can be used to catalyze hydroxylation reactions described above.

Known enzyme families with terminal hydroxylation activity for medium and long chain alkanes and fatty acids include AlkB, CYP52, CYP153, and LadA (Bordeaux et al., 2012, Angew. Chem.-Int. Edit. 51: 10712-10723; Ji et al., 2013, Front. Microbiol. 4). For example, Malca et al. describe terminal hydroxylation of mono-unsaturated fatty acid by cytochromes P450 of the CYP153 family (Malca et al., 2012, Chemical Communications 48: 5115-5117). Weissbart et al. describe the terminal hydroxylation of various cis and trans unsaturated lauric acid analogs (Weissbart et al., 1992, Biochimica et Biophysica Acta, Lipids and Lipid Metabolism 1124: 135-142). However, to date, none of these enzymes has been demonstrated to perform terminal hydroxylation of alkenes with internal olefins such as (E)-dec-5-ene. The presence of C═C bonds present competing sites of oxygen insertion and alters the 3-dimensional orientation of the molecule. The regioselectivity of these enzymes for the terminal C—H bond of alkanes and fatty acid substrate may not extend to alkenes with internal olefins for these reasons. For asymmetric substrates, obtaining hydroxylation at the desired terminal C—H bond presents additional challenges compared to symmetric substrates. Finally, controlling the reaction selectivity to produce a single terminal alcohol instead of α-ω diols, acids, or diacids is also a major concern.

In particular embodiments, the search for a terminal hydroxylase with activity for alkene with internal olefins starts with known terminal alkane and fatty acid hydroxylases. There are four families of enzymes with reported terminal alkane and fatty acid hydroxylation activity: (1) methane monoxxygenases; (2) integral membrane diiron non-heme alkane hydroxylases (AlkB); (3) Cytochrome P450s (P450s); and (4) long chain alkane monoxygenases (LadA) (Bordeaux et al., 2012, Angew. Chem.-Int. Edit. 51: 10712-10723; Ji et al., 2013, Front. Microbiol. 4). Methane monooxygenases are difficult to express in heterologous non-methanotrophic hosts and generally prefer small substrate (<C4). Of the remaining three families, the substrate specificity based on substrate chain length of representative members is summarized below in Table 2.

TABLE 2 Relative activities of terminal hydroxylases for alkanes and fatty acids with various chain lengths. LadA CYP52 CYP52 CYP15 AlkB (Feng et A3 A4 CYP52 CYP15 3A P. P. putida alkB2 al., (Scheller (Scheller A21 CYP153 3A16 sp. GPo1 Gordonia 2007, et al., et al., (Kim et A6 (Scheps et (Scheps et (Vanbeilen sp TF6* Proc. 1996, 1996, al., 2007, (Funhoff al., 2011, al., 2011, et al., 1994, (Fujii et al., Natl. Arch. Arch. Arch. et al., Org. Org. Enzyme 2004, Biosci. Acad. Biochem Biochem Biochem. 2006, J. Biomol. Biomol. Microb. Biotechnol. Sci. U.S.A. Biophys. Biophys. Biophys. Alkane/FA Bacteriol. Chem. 9: Chem. 9: Technol. 16: Biochem. 68: 104: 328: 328: 464: chain length 188: 5220-5227) 6727-6733) 6727-6733) 904-911) 2171-2177) 5602-5607) 245-254) 245-254) 213-220) C8 100 100 100 95 72 C9 82 29 69 100 63 C10 23 13 60 60 66 C11 1 <8 <6 6 48 C12 34 41 37 C12 FA 20 100 100 (lauric) C14 C14 FA 86 (Myristic) C15 83 C16 100 100 33 C16 FA 35 18 29 (Palmetic) C18 78 48 20 C18 FA 30 1 (Stearic) C22 74 C24 65 *100% relative activity obtained with hexane

In certain embodiments, depending on the chain length of the desired substrate, some members of these four enzyme families are better suited than others as candidates for evaluation. For C-10 substrates such as (E)-dec-5-ene, the substrate specificity of characterized CYP153 and AlkB enzymes makes them candidate enzymes. Likewise, for longer substrates such as (Z)-hexadec-11-ene, members of the LadA and CYP52 families appear to have the closest substrate profile.

The most widely characterized member of the AlkB family is obtained from the Alk system of Pseudomonas putida GPo1 (van Beilen and Funhoff, 2005, Curr. Opin. Biotechnol. 16: 308-314). In addition to the integral membrane diiron non-heme hydroxylase AlkB, a rubredoxin (AlkG) and a rubredoxin reductase (AlkT) are required for hydroxylation function. The entire Alk system of P. putida GPo1, alkBFGHJKL and alkST genes, which allows the strain to grown on alkanes as its sole carbon source, has been cloned into the broad host range vector pLAFR1 (pGEc47) and is available from DSMZ in the host E. Coli K12 Gec137 (Smits et al., 2001, Plasmid 46: 16-24). The other alk genes alkF, alkJ, alkH, alkK, alkL, and alkS encode an inactive rubredoxin, an alcohol dehydrogenase, an aldehyde dehydrogenase, an acyl-CoA synthase, an alkane transporter and a global pathway regulator, respectively (Smits et al., 2003, Antonie Van Leeuwenhoek 84: 193-200). These genes facilitate the use of the alcohol product from the AlkB reaction to generate the fatty acyl-CoA that is substrate for β-oxidation. To accumulate the alcohol product, a knockout strain of alkJ, E. coli GEC137 pGEc47ΔJ has been used in a whole-cell biotransformation to produce 1-dodecanol (Grant et al., 2011, Enzyme Microb. Technol. 48: 480-486). The presence of alkL appears to enhance substrate uptake and consequently improve the whole-cell activity for both Pseudomonas and E. coli (Cornelissen et al., 2013, Biotechnology and Bioengineering 110: 1282-1292; Julsing et al., 2012, Appl. Environ. Microbiol. 78: 5724-5733; Scheps et al., 2013, Microb. Biotechnol. 6: 694-707). A simplified version of pGEc47 containing only alkBFGST in the broad-host range vector pCOM10, pBT10, has also been used for the conversion of fatty-acid methyl esters to w-hydroxy fatty acid methyl esters in E. coli W3110 (Schrewe et al., 2011, Advanced Synthesis & Catalysis 353: 3485-3495).

CYP52 family members are membrane bound cytochrome P450s that require electron delivery from a reductase for function. CYP52 members have mainly been identified from alkane-degrading Candida species (Scheller et al., 1996, Arch. Biochem. Biophys. 328: 245-254; Craft et al., 2003, Appl. Environ. Microbiol. 69: 5983-5991; Scheller et al., 1998, J. Biol. Chem. 273: 32528-32534; Seghezzi et al., 1992, DNA Cell Biol. 11: 767-780; Zimmer et al., 1996, Biochem. Biophys. Res. Commun. 224: 784-789). Thus far, expression and characterization of CYP52 enzymes have been performed in the native Candida host and other yeast hosts. Gene knockouts of (1) the β-oxidation pathways, (2) alcohol dehydrogenases and (3) select native CYP52s has resulted in strains that can accumulate ω-hydroxy fatty acids when fatty acids are fed to the culture (Lu et al., 2010, J. Am. Chem. Soc. 132: 15451-15455). Of particular interest, DP428, DP522 and DP526 are C. tropicalis strains expressing a single CYP52 with the appropriate knockouts for catalyzing terminal hydroxylation of fatty acids (Lu et al., 2010, J. Am. Chem. Soc. 132: 15451-15455).

CYP153 family members are soluble and membrane associated cytochrome P450s that also depend on electron transfer from ferredoxin and ferredoxin reductase for function (Funhoff et al., 2007, Enzyme and Microbial Technology 40: 806-812). CYP153 members have been isolated from a range of alkane-degrading microorganisms. There are currently 56 annotated CYP153 sequences available from the Nelson P450 database, a BLAST search of CYP153A6 resulted in 221 identified homologs with >70% sequence identity. The use of CYP153 enzymes for terminal hydroxylation of octane and dodecanoic acid has been demonstrated with heterologous expression in E. coli. For the conversion of octane to octanol, the CYP153 operon from Mycobacterium sp. HXN-1500 was cloned into pET28b(+) and the biotransformation was performed in E. coli BL21(DE3) (Gudiminchi et al., 2012, Appl. Microbial. Biotechnol. 96: 1507-1516). For the conversion of dodecanoic acid, an E. coli HMS174 strain containing a fusion of a CYP153A_(Maq). mutant with the CYP102A1 reductase domain in pColaDuet-1 along with alkL was used for the transformation (Scheps et al., 2013, Microb. Biotechnol. 6: 694-707).

Long chain alkane monooxygenase, LadA, isolated from G. thermodenitrificants NG80-2 catalyzes the terminal hydroxylation of C15 to C36 alkanes with a metal-free flavoprotein mechanism that differs from AlkB and CYP enzymes (Dong et al., 2012, Appl. Microbiol. Biotechnol. 94: 1019-1029). The LadA reaction requires FMNH₂ or NADPH and the native reductase partner has yet to be identified. Expression of the LadA gene in E. coli BL21 (DE3) using the pET-28a(+) plasmid yielded cell extracts with terminal hydroxylation activity for hexadecane (Dong et al., 2012, Appl. Microbial. Biotechnol. 94: 1019-1029). Literature reports of LadA hydroxylation reactions have been performed using purified enzymes and examples of whole-cell biotransformation is lacking.

Coding sequences for enzymes that may be used herein may be derived from bacterial, fungal, or plant sources. Tables 3, 4, and 5 list enzymes for coding regions of representative non-heme diiron alkane monooxygenases, long-chain alkane hydroxylases, and cytochromes P450, respectively. Additional enzymes and their coding sequences may be identified by BLAST searching of public databases. Typically, BLAST searching of publicly available databases with known non-heme diiron alkane monooxygenases, cytochromes P450, and long-chain alkane hydroxylase sequences, such as those provided herein, is used to identify enzymes and their encoding sequences that may be used in the present invention. For example, enzymes having amino acid sequence identities of at least about 80-85%, 85%-90%, 90%-95%, or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the enzymes listed in Tables 3, 4, and 5 may be used. Hydroxylase enzymes can be codon-optimized for expression in certain desirable host organisms, such as yeast and E. coli.

In other embodiments, the sequences of the enzymes provided herein may be used to identify other homologs in nature. For example, each of the encoding nucleic acid fragments described herein may be used to isolate genes encoding homologous proteins. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to, (1) methods of nucleic acid hybridization, (2) methods of DNA and RNA amplification, as exemplified by various uses of nucleic acid amplification technologies (e.g., polymerase chain reaction (PCR), Mullis et al., U.S. Pat. No. 4,683,202; ligase chain reaction (LCR), Tabor, S. et al., Proc. Acad. Sci. USA 82:1074 (1985); or strand displacement amplification (SDA), Walker et al., Proc. Natl. Acad. Sci. USA, 89:392 (1992)), and (3) methods of library construction and screening by complementation.

Hydroxylase enzymes or whole cells expressing hydroxylase enzymes can be further engineered for use in the methods of the invention. Enzymes can be engineered for improved hydroxylation activity, improved Z:E selectivity, improved regioselectivity, improved selectivity for hydroxylation over epoxidation and/or improved selectivity for hydroxylation over dehalogenation. The term “improved hydroxylation activity” as used herein with respect to a particular enzymatic activity refers to a higher level of enzymatic activity than that measured in a comparable non-engineered hydroxylase enzyme of whole cells comprising a hydroxylase enzyme. For example, overexpression of a specific enzyme can lead to an increased level of activity in the cells for that enzyme. Mutations can be introduced into a hydroxylase enzyme resulting in engineered enzymes with improved hydroxylation activity. Methods to increase enzymatic activity are known to those skilled in the art. Such techniques can include increasing the expression of the enzyme by increasing plasmid copy number and/or use of a stronger promoter and/or use of activating riboswitches, introduction of mutations to relieve negative regulation of the enzyme, introduction of specific mutations to increase specific activity and/or decrease the K_(M) for the substrate, or by directed evolution. See, e.g., Methods in Molecular Biology (vol. 231), ed. Arnold and Georgiou, Humana Press (2003).

Accordingly, some embodiments of the invention provide methods for synthesizing olefinic alcohol products as described above, wherein the enzyme is a non-heme diiron monooxygenase. In some embodiments, the non-heme diiron monooxygenase is selected from Table 3 or a variant thereof having at least 90% identity thereto.

TABLE 3 Non-heme diiron monooxygenase enzymes capable of catalyzing selective terminal alkene hydroxylation. Species Origin Gene Name Accession No Pseudomonas oleovorans alkB P12691 Pseudomonas mendocina (strain ymp) Pmen_0443 A4XPE8 Pseudomonas aeruginosa alkB Q932R7 Enterobacteriaceae bacterium 58 alkB B5TVB4 Bacillus sp. BTRH40 alkB B5TVB3 uncultured bacterium alkB B6Z2G6 Pseudomonas aeruginosa alk B7U6M1 uncultured bacterium alkB U3PXQ1 uncultured bacterium alkB U3Q1X4 Pseudomonas stutzeri (Pseudomonas perfectomarina) alkB Q7X4G8 uncultured organism alkB G3EBX4 uncultured bacterium alkB U3PXQ7 Pseudomonas aeruginosa alk B7U6M0 Pseudomonas chlororaphis subsp. aureofaciens alkB Q9RLI5 Arthrobacter sp. ITRH48 alkB B5TVB7 Streptomyces sp. ITRH51 alkB B5TVB6 Arthrobacter sp. ITRH49 alkB B5TVC0 Dietzia sp. ITRH56 alkB B5TVB8 Microbacterium sp. ITRH47 alkB B5TVB5 Pantoea sp. BTRH11 alkB B5TVB2 Pseudomonas sp. ITRI53 alkB B5TVB1 Pseudomonas sp. ITRI73 alkB B5TVB0 Pseudomonas sp. ITRH25 alkB B5TVA9 Pseudomonas sp. MIXRI75 alkB B5TVA8 Pseudomonas sp. MIXRI74 alkB B5TVA7 Rhodococcus sp. ITRH43 alkB B5TVA4 Ochrobactrum sp. ITRH1 alkB B5TVA3 Alcaligenaceae bacterium BTRH5 alkB B5TVA6 Pseudomonas sp. ITRH76 alkB B5TVA5 Pseudomonas sp. 7/156 alkB Q93LR8 uncultured Rhizobiales bacterium alkB D6NSH3 uncultured soil bacterium S5DSW0 uncultured bacterium alkB U3PYH2 uncultured prokaryote alkB C7EAT4 uncultured Rhizobiales bacterium alkB D6NSL1 uncultured Rhizobiales bacterium alkB D6NSJ4 uncultured prokaryote alkB C7EAZ5 uncultured Rhizobiales bacterium alkB D6NSK5 uncultured Rhizobiales bacterium alkB D6NSK3 uncultured Rhizobiales bacterium alkB D6NSJ7 uncultured Rhizobiales bacterium alkB D6NSK1 uncultured Rhizobiales bacterium alkB D6NSH4 uncultured Rhizobiales bacterium alkB D6NSJ2 uncultured Rhizobiales bacterium alkB D6NSI2 uncultured Rhizobiales bacterium alkB D6NSJ3 uncultured Rhizobiales bacterium alkB D6NSJ6 Pseudomonas sp. ITRI22 alkB B5TVB9 uncultured Rhizobiales bacterium alkB D6NSK7 uncultured soil bacterium S5DTG4 Pseudomonas putida (Arthrobacter siderocapsulatus) alkB Q9WWW6 uncultured Rhizobiales bacterium alkB D6NSI6 uncultured bacterium alkB B6Z2E6 uncultured bacterium alkB B1P6K4 Pseudomonas sp. G5(2012) PG5_40690 S2EW96 Alcanivorax dieselolei alkB B6Z2B7 Alcanivorax borkumensis alkB B6Z284 uncultured bacterium alkB B6Z2G9 Marinobacter sp. S17-4 alkB C7DLJ8 uncultured bacterium alkB B6Z2H0 Alcanivorax sp. S17-16 alkB B6Z2D8 uncultured organism alkB G3EBX7 uncultured bacterium alkB H9NJ23 uncultured bacterium alkB C8AYB7 uncultured bacterium alkB W0UB63 uncultured bacterium alkB U3Q1V0 Alcanivorax borkumensis alkB T1WPB9 uncultured organism alkB G3EBX5 uncultured Rhizobiales bacterium alkB D6NSK6 uncultured bacterium alkB U3Q5C8 uncultured bacterium alkB Q3HXE5 Xanthobacter flavus alkane-1-monooxygenase Q934J9 uncultured bacterium alkB Q3HXD6 Acidisphaera sp. C197 alkB Q5RLH8 uncultured bacterium alkB M9T624 uncultured bacterium alkB M9T8D1 uncultured bacterium alkB H9B8U8 Kordiimonas gwangyangensis alkB B6Z2E4 uncultured soil bacterium S5DPL2 uncultured bacterium alkB F0X332 uncultured bacterium alkB F0X324 uncultured bacterium alkB F0X334 uncultured organism alkB G3EBX2 uncultured bacterium alkB F0X328 uncultured soil bacterium S5DTI7 uncultured bacterium alkB Q3HXF7 uncultured bacterium alkB F0X327 uncultured bacterium alkB F0X335 uncultured bacterium alkB F0X329 uncultured bacterium alkB F0X342 uncultured bacterium alkB F0X300 uncultured bacterium alkB Q3HXE8 uncultured bacterium alkB U3Q1X0 uncultured bacterium alkB Q3HXD7 Ralstonia sp. PT11 alkB Q3HXC9 uncultured bacterium alkB Q3HXE6 uncultured bacterium alkB F0X305 uncultured bacterium alkB U3Q5A0 uncultured bacterium alkB F0X306 Marinobacter sp. P1-14D alkB1 C6KEH4 uncultured Rhizobiales bacterium alkB D6NSI7 uncultured bacterium alkB F0X346 uncultured bacterium alkB F0X346 uncultured bacterium alkB F0X343 uncultured bacterium alkB F0X339 uncultured bacterium alkB F0X309 uncultured bacterium alkB F0X333 uncultured bacterium alkB F0X321 uncultured bacterium alkB Q3HXF0 uncultured bacterium alkB F0X312 uncultured bacterium alkB F0X303 uncultured bacterium alkB F0X331 uncultured bacterium alkB F0X302 uncultured bacterium alkB Q3HXE9 uncultured bacterium alkB F0X313 uncultured bacterium alkB F0X316 uncultured bacterium alkB M9TDK6 uncultured bacterium alkB H9B8V5 uncultured Rhizobiales bacterium alkB D6NSF4 uncultured Rhizobiales bacterium alkB D6NSF2 uncultured bacterium alkB B6Z2G8 uncultured Rhizobiales bacterium alkB D6NSF1 uncultured Rhizobiales bacterium alkB D6NSG4 uncultured Rhizobiales bacterium alkB D6NSG3 uncultured Rhizobiales bacterium alkB D6NSF3 uncultured Rhizobiales bacterium alkB D6NSI4 uncultured Rhizobiales bacterium alkB D6NSH9 uncultured Rhizobiales bacterium alkB D6NSG1 uncultured Rhizobiales bacterium alkB D6NSJ9 uncultured Rhizobiales bacterium alkB D6NSG6 uncultured soil bacterium S5DP42 uncultured bacterium alkB F0X323 uncultured bacterium alkB F0X318 uncultured bacterium alkB F0X317 uncultured bacterium alkB F0X325 uncultured bacterium alkB F0X308 uncultured bacterium alkB F0X336 uncultured soil bacterium S5E0W0 uncultured bacterium alkB F0X304 Bradyrhizobium sp. DFCI-1 C207_00091 U1HQ84 uncultured Rhizobiales bacterium alkB D6NSF9 uncultured Rhizobiales bacterium alkB D6NSH2 uncultured Rhizobiales bacterium alkB D6NSF6 uncultured Rhizobiales bacterium alkB D6NSG2 uncultured Rhizobiales bacterium alkB D6NSH7 uncultured bacterium alkB F0X322 uncultured soil bacterium S5DPY4 uncultured bacterium alkB F0X349 uncultured bacterium alkB F0X310 uncultured bacterium alkB F0X315 uncultured bacterium alkB F0X344 uncultured bacterium alkB F0X326 uncultured bacterium alkB W0UB94 uncultured bacterium alkB W0UAL7 uncultured soil bacterium S5DP84 uncultured soil bacterium S5E064 uncultured soil bacterium S5E0M5 uncultured bacterium alkB M9T7Y4 uncultured prokaryote alkB C7EAZ7 Thalassolituus oleivorans alkB Q8RSS6 uncultured prokaryote alkB C7EAZ8 Marinobacter sp. EVN1 Q672_13115 U7NVU4 uncultured Rhizobiales bacterium alkB D6NSF8 Marinobacter aquaeolei (strain ATCC 700491/DSM Maqu_0610 A1TY92 11845/VT8) (Marinobacter hydrocarbonoclasticus (strain DSM 11845)) Marinobacter hydrocarbonoclasticus ATCC 49840 alkB MARHY2847 H8WCU7 uncultured Rhizobiales bacterium alkB D6NSG8 Alcanivorax borkumensis alkB1 Q93UQ1 Alcanivorax borkumensis (strain SK2/ATCC alkB1 ABO_2707 Q0VKZ3 700651/DSM 11573) Marinobacter aquaeolei (strain ATCC 700491/DSM Maqu_0440 A1TXS2 11845/VT8) (Marinobacter hydrocarbonoclasticus (strain DSM 11845)) Alcanivorax sp. 97CO-5 Y017_07510 W7AC06 Marinobacter sp. C1S70 Q667_13505 U7P171 Marinobacter sp. EVN1 Q672_13130 U7NYF9 Pseudoxanthomonas spadix (strain BD-a59) DSC_08960 G7UVX3 Marinobacter sp. EN3 Q673_04890 U7H9M7 Marinobacter sp. ES-1 Q666_09550 U7G9A6 Oceanicaulis sp. HTCC2633 OA2633_08724 A3UHL2 Citreicella sp. 357 C357_19621 I1AS33 Caulobacter sp. (strain K31) Caul_5439 B0TA04 Thalassolituus oleivorans MIL-1 TOL_1423 M5DQR5 uncultured bacterium alkB W0UAQ4 uncultured bacterium alkB W0UAL9 uncultured bacterium alkB W0UAQ9 gamma proteobacterium NOR5-3 NOR53_3428 B8KLY6 uncultured marine microorganism 21G8-5 A5CFX9 uncultured marine microorganism 9E7-8 A5CFU5 Alcanivorax pacificus W11-5 S7S_02132 K2GLA3 Alcanivorax dieselolei C3W4W7 Alcanivorax sp. PN-3 Q668_06955 U7I1M1 Alcanivorax dieselolei (strain DSM 16502/CGMCC B5T_00721 K0C8Z6 1.3690/B-5) Alcanivorax dieselolei alkB2 D2JNY2 bacterium enrichment culture clone US3-MTBE mdpA L7T214 bacterium enrichment culture clone US2-MTBE mdpA L7SZY0 Marinobacter sp. ELB17 MELB17_10558 A3JHB9 Marinobacter sp. BSs20148 alkB1 MRBBS_1602 M1FBW8 Pseudomonas alcaligenes NBRC 14159 alkB PA6_005_01830 U3AUD1 Simiduia agarivorans (strain DSM 21679/JCM 13881/ M5M_18065 K4KP06 BCRC 17597/SA1) gamma proteobacterium HTCC2207 GB2207_03060 Q1YPC4 Limnobacter sp. MED105 LMED105_14555 A6GTF8 Alcanivorax sp. R8-12 alkB2 R9R6I2 Gammaproteobacteria bacterium MOLA455 alkB1 U062_00014 W2UFM4 Alcanivorax hongdengensis A-11-3 A11A3_01150 L0WGR7 Acidovorax sp. KKS102 C380_12125 K0IAD8 Moritella sp. PE36 PE36_11657 A6FHH9 Moritella sp. PE36 PE36_11657 A6FHH9 Ahrensia sp. R2A130 alkB R2A130_3229 E0MP68 Hoeflea phototrophica DFL-43 HPDFL43_04645 A9D3P4 Curvibacter putative symbiont of alkB Csp_A02180 C9Y7W7 Hydra magnipapillata Pseudovibrio sp. JE062 PJE062_1512 B6QXF8 Oxalobacteraceae bacterium IMCC9480 IMCC9480_2292 F1W4Y4 Methylibium petroleiphilum (strain PM1) alkB Mpe_B0606 A2SP81 Ralstonia sp. AU12-08 C404_01360 S9TME3 Burkholderia phytofirmans (strain DSM 17436/PsJN) Bphyt_5401 B2TBV7 gamma proteobacterium BDW918 DOK_05250 I2JMD3 Pseudovibrio sp. (strain FO-BEG1) alkB PSE_3490 G8PKM2 Bradyrhizobium sp. DFCI-1 C207_06028 U1H8I8 Alcanivorax dieselolei (strain DSM 16502/CGMCC B5T_04393 K0CLJ4 1.3690/B-5) Alcanivorax sp. PN-3 Q668_04650 U7HLN0 Alcanivorax dieselolei alkB1 Q6B431 Burkholderia thailandensis E444 BTJ_212 W6C501 Burkholderia thailandensis 2002721723 BTQ_2100 W6BLA1 Burkholderia thailandensis H0587 BTL_1506 W6BA85 Burkholderia thailandensis (strain E264/ BTH_I1814 Q2SXK3 ATCC 700388/DSM 13276/CIP 106301) Burkholderia pseudomallei 1026b BP1026B_I0975 I1WH83 Burkholderia pseudomallei 1026a BP1026A_4019 I2KNJ5 Burkholderia pseudomallei MSHR305 BDL_3139 S5P5X7 Burkholderia pseudomallei 305 alkB BURPS305_7408 A4LDP5 Burkholderia pseudomallei Pasteur 52237 alkB BURPSPAST_R0133 A8KVJ2 Burkholderia pseudomallei (strain K96243) BPSL2350 Q63SH1 Burkholderia pseudomallei (strain 1710b) BURPS1710b_2801 Q3JQG8 Burkholderia pseudomallei BPC006 BPC006_I2776 K7Q7Y2 Burkholderia pseudomallei 1710a alkB BURPS1710A_3234 C6TUD4 Burkholderia pseudomallei 1106b alkB_2BURPS1106B_A1957 C5ZKC8 Burkholderia pseudomallei (strain 1106a) alkB BURPS1106A_2735 A3NXB5 Burkholderia pseudomallei (strain 668) BURPS668_2678 A3NBI1 Burkholderia pseudomallei NCTC 13178 BBJ_481 V9Y591 Burkholderia pseudomallei MSHR1043 D512_14116 M7EHA3 Burkholderia pseudomallei 354a BP354A_0895 I2MQ94 Burkholderia pseudomallei 354e BP354E_0708 I2MD23 Burkholderia pseudomallei 1258b BP1258B_0905 I2LQQ4 Burkholderia pseudomallei 1258a BP1258A_0812 I2LKD3 Burkholderia pseudomallei 576 alkB BUC_2998 B7CM79 Burkholderia pseudomallei 1655 alkB BURPS1655_H0133 B2HAC8 Burkholderia pseudomallei S13 alkB BURPSS13_V0139 B1HDJ2 Burkholderia pseudomallei 406e alkB BURPS406E_H0229 A8EBS1 Burkholderia pseudomallei MSHR146 BBN_1088 W0PXC8 Burkholderia pseudomallei MSHR511 BBQ_961 W0MCN0 Burkholderia pseudomallei NAU20B-16 BBS_2570 V9YGA1 Burkholderia pseudomallei MSHR346 GBP346_A2857 C4KQU6 Burkholderia pseudomallei MSHR338 M218_13015 W1M8G5 Burkholderia xenovorans (strain LB400) Bxe_B1208 Q13ME1 Burkholderia thailandensis MSMB43 A33K_14899 I6AHY8 Burkholderia sp. Ch1-1 BCh11DRAFT_02054 I2IU52 Alcanivorax sp. R8-12 alkB3 R9R6Q8 gamma proteobacterium HTCC5015 GP5015_636 B5JV27 Alcanivorax pacificus W11-5 S7S_03034 K2GFU4 Actinoplanes sp. (strain ATCC 31044/CBS 674.73/ alkB ACPL_4910 G8SLX8 SE50/110) Alcanivorax sp. DG881 ADG881_1174 B4X426 Methylibium sp. T29-B alkB1 Y694_03823 W7WAG2 Methylibium sp. T29 mdpA X551_03232 W7VT91 Burkholderia thailandensis MSMB121 BTI_1284 N0AI18 Burkholderia sp. TJI49 B1M_44170 F0GKQ0 Burkholderia mallei (strain ATCC 23344) alkB BMA0635 Q62LK2 Burkholderia mallei (strain NCTC 10247) alkB BMA10247_1692 A3MLU7 Burkholderia mallei (strain NCTC 10229) alkB BMA10229_A2910 A2SA87 Burkholderia mallei (strain SAVP1) alkB BMASAVP1_A2377 A1V630 Burkholderia mallei PRL-20 alkB BMAPRL20_A0647 C5NLY3 Burkholderia mallei GB8 horse 4 BMAGB8_0674 C4AYJ3 Burkholderia mallei ATCC 10399 alkB BMA10399_E0136 A9KA35 Burkholderia mallei JHU alkB BMAJHU_C0140 A5XN41 Burkholderia mallei FMH alkB BMAFMH_C0136 A5XJ42 Burkholderia mallei 2002721280 alkB BMA721280_A1345 A5TJ65 Burkholderia pseudomallei Pakistan 9 alkB BUH_2787 C0YFB6 Burkholderia sp. (strain 383) (Burkholderia cepacia Bcep18194_A4085 Q39IN4 (strain ATCC 17760/NCIB 9086/R18194)) Ralstonia sp. 5_2_56FAA HMPREF0989_00681 U3G9A8 Ralstonia sp. 5_7_47FAA HMPREF1004_00261 E2ST40 Burkholderia cenocepacia (strain AU 1054) Bcen_0501 Q1BY92 Burkholderia cenocepacia (strain HI2424) Bcen2424_0980 A0K5F6 Burkholderia sp. KJ006 MYA_0870 I2DKR1 Burkholderia vietnamiensis (strain G4/LMG 22486) Bcep1808_0897 A4JCA5 (Burkholderia cepacia (strain R1808)) Burkholderia cenocepacia KC-01 P355_2107 V5A0K9 Ralstonia pickettii (strain 12D) Rpic12D_4221 C6BN09 Ralstonia pickettii (strain 12J) Rpic_4109 B2UI09 Ralstonia pickettii OR214 OR214_00862 R0CSD0 Mycobacterium thermoresistibile ATCC 19527 KEK_22639 G7CND0 Burkholderia cenocepacia PC184 BCPG_00786 A2VS55 Parvularcula bermudensis (strain ATCC BAA-594/ PB2503_09204 E0TD71 HTCC2503/KCTC 12087) Rhodococcus triatomae BKS 15-14 G419_20650 M2WXQ1 Alcanivorax hongdengensis A-11-3 A11A3_01155 L0WH65 Alcanivorax hongdengensis G1C7G7 Micromonospora sp. ATCC 39149 MCAG_04553 C4REI2 Micromonospora lupini str. Lupac 08 alkB MILUP08_41795 I0KZ81 Patulibacter medicamentivorans PAI11_23570 H0E6A7 Burkholderia cenocepacia (strain ATCC BAA-245/DSM BCAL3029 B4EBR3 16553/LMG 16656/NCTC 13227/J2315/CF5610) (Burkholderia cepacia (strain J2315)) Burkholderia cenocepacia BC7 BURCENBC7_AP5666 U1ZCU6 Burkholderia cenocepacia K56-2Valvano BURCENK562V_C5856 T0E860 Burkholderia cenocepacia H111 I35_3695 G7HIJ0 Burkholderia cepacia GG4 GEM_2548 J7J4L5 Burkholderia ambifaria IOP40-10 BamIOP4010DRAFT_1629 B1FC70 Burkholderia vietnamiensis AU4i L810_3738 U2H0D0 Burkholderia ambifaria MEX-5 BamMEX5DRAFT_0109 B1SX43 Burkholderia cenocepacia (strain MC0-3) Bcenmc03_0941 B1JX99 Burkholderia cepacia (Pseudomonas cepacia) alkB Q9AEN3 Burkholderia multivorans CGD1 BURMUCGD1_2488 B9BAK1 Burkholderia multivorans (strain ATCC 17616/249) alkB BMULJ_00816 B3CYB3 Burkholderia multivorans (strain ATCC 17616/249) alkB BMULJ_00816 B3CYB3 Burkholderia multivorans CGD2M BURMUCGD2M_2894 B9CFY2 Burkholderia multivorans CGD2 BURMUCGD2_2807 B9BSN6 Burkholderia glumae (strain BGR1) bglu_1g25240 C5AA12 Burkholderia multivorans CF2 BURMUCF2_0698 J5AST2 Burkholderia multivorans ATCC BAA-247 BURMUCF1_0763 J4JJJ2 Mycobacterium xenopi RIVM700367 MXEN_06581 I0RWI2 Alcanivorax sp. P2S70 Q670_07625 U7G3V1 Rhodococcus sp. p52 alkB U5S015 Rhodococcus pyridinivorans AK37 AK37_15478 H0JTS8 Micromonospora sp. M42 MCBG_00051 W7V9N0 Nocardia nova SH22a NONO_c63170 W5TPA6 Actinoplanes missouriensis (strain ATCC 14538/DSM AMIS_28610 I0H4Z4 43046/CBS 188.64/JCM 3121/NCIMB 12654/ NBRC 102363/431) Mycobacterium thermoresistibile ATCC 19527 KEK_04707 G7CD93 Streptomyces collinus Tu 365 B446_00650 B446_34640 S5VEV9 Mycobacterium smegmatis MKD8 alkB D806_1894 L8FH78 Mycobacterium smegmatis (strain ATCC 700084/ alkB MSMEG_1839 A0QTH1 mc(2)155) MSMEI_1797 Burkholderia gladioli (strain BSR3) bgla_1g28520 F2LCU4 Nocardia cyriacigeorgica (strain GUH-2) alkB NOCYR_2725 H6R6Y1 Mycobacterium sp. (strain Spyr1) Mspyr1_40540 E6TPD9 Mycobacterium gilvum (strain PYR-GCK) Mflv_4721 A4TF88 (Mycobacterium flavescens (strain ATCC 700033/ PYR-GCK)) Mycobacterium hassiacum DSM 44199 C731_1322 K5BKD8 Mycobacterium phlei RIVM601174 MPHLEI_02293 I0S2Q3 Burkholderia ambifaria (strain MC40-6) BamMC406_0853 B1YUL7 Conexibacter woesei (strain DSM 14684/JCM 11494/ Cwoe_5739 D3F1V9 NBRC 100937/ID131577) Burkholderia ambifaria (strain ATCC BAA-244/ Bamb_0841 Q0BHH3 AMMD) (Burkholderia cepacia (strain AMMD)) Mycobacterium vaccae ATCC 25954 MVAC_06502 K0V939 Streptomyces sp. AA4 SSMG_06597 D9UYP9 Nocardia asteroides NBRC 15531 alkB NCAST_33_00580 U5EK43 Hydrocarboniphaga effusa AP103 WQQ_35830 I8T3V4 Mycobacterium sp. (strain Spyr1) Mspyr1_27000 E6TM45 Rhodococcus sp. EsD8 EBESD8_14280 N1M251 Rhodococcus pyridinivorans SB3094 Y013_10875 Y013_14995 V9XCI1 uncultured bacterium alk A7XY59 Dietzia sp. D5 W0C8S6 Gordonia amarae NBRC 15530 alkB GOAMR_34_00200 G7GP29 gamma proteobacterium BDW918 DOK_15269 I2JH75 Marinobacter sp. EVN1 Q672_03155 U7NQ32 Marinobacter santoriniensis NKSG1 MSNKSG1_09613 M7CV98 Marinobacter sp. ES-1 Q666_05770 U7GFG6 gamma proteobacterium HdN1 alkM HDN1F_04190 E1VGR0 Nocardia farcinica (strain IFM 10152) NFA_33210 Q5YUH3 Mycobacterium chubuense (strain NBB4) Mycch_2783 I4BJT7 Acinetobacter towneri DSM 14962 = CIP 107472 F947_01315 N9CH84 Rhodococcus erythropolis CCM2595 O5Y_10330 T1VNI2 Rhodococcus erythropolis (strain PR4/NBRC 100887) alkB RER_21620 C0ZWY5 Rhodococcus sp. P27 N806_20680 U0E9X4 Rhodococcus erythropolis DN1 N601_09550 T5IBP7 Rhodococcus erythropolis (Arthrobacter picolinophilus) alkB A4ZZL2 Mycobacterium fortuitum subsp. fortuitum DSM 46621 MFORT_07571 K0VIS2 Rhodococcus qingshengii BKS 20-40 G418_14624 M2XAS5 Rhodococcus erythropolis (Arthrobacter picolinophilus) alkB2 Q9AE68 Rhodococcus sp. (strain RHA1) alkB RHA1_ro02534 Q0SDP7 Rhodococcus sp. JVH1 JVH1_3134 J1RMD5 Rhodococcus wratislaviensis IFP 2016 Rwratislav_18854 L2TK91 Rhodococcus wratislaviensis alkB1 K7WI49 Rhodococcus sp. (strain Q15) alkB2 Q93DM7 Rhodococcus opacus M213 WSS_A20069 K8XV97 Rhodococcus erythropolis (Arthrobacter picolinophilus) alkB V5LET8 Streptomyces sp. AA4 SSMG_06805 D9V1L5 Geobacillus sp. MH-1 alkB-geo6 C5J0F7 Mycobacterium neoaurum VKM Ac-1815D D174_08465 V5X9E7 Rhodococcus imtechensis RKJ300 = JCM 13270 W59_13161 I0WSJ7 Prauserella rugosa alkB Q9XBM1 Rhodococcus erythropolis SK121 RHOER0001_4201 C3JG64 Amycolatopsis azurea DSM 43854 C791_5134 M2PZK0 Mycobacterium rhodesiae (strain NBB3) MycrhN_0412 G8RK27 Rhodococcus ruber alkB7 D3U111 Rhodococcus ruber BKS 20-38 G352_25762 M2XQQ3 Mycobacterium chubuense (strain NBB4) D2JYT1 Mycobacterium chubuense (strain NBB4) Mycch_1351 I4BFU6 Mycobacterium smegmatis JS623 Mycsm_01384 L0IUF4 Nocardia nova SH22a alkB NONO_c46180 W5TJL9 Rhodococcus sp. BCP1 alkB E5G6V9 Saccharomonospora marina XMU15 SacmaDRAFT_4417 H5X9W5 Mycobacterium sp. (strain JLS) Mjls_1369 A3PW94 Rhodococcus ruber alkB7 D3U119 Mycobacterium tuberculosis BT1 alkB HKBT1_3428 W6HJ76 Mycobacterium tuberculosis BT2 alkB HKBT2_3435 W6H3Z6 Mycobacterium tuberculosis HKBS1 alkB HKBS1_3438 W6GVB7 Mycobacterium tuberculosis EAI5 M943_16800 S5F023 Mycobacterium tuberculosis EAI5/NITR206 J114_17435 R4MLW1 Mycobacterium tuberculosis CAS/NITR204 J113_22685 R4MIF7 Mycobacterium bovis (strain ATCC BAA-935/ alkB Mb3280c Q7TWW3 AF2122/97) Mycobacterium tuberculosis (strain ATCC 25618/ alkB Rv3252c RVBD_3252c O05895 H37Rv) Mycobacterium tuberculosis str. Beijing/NITR203 J112_17475 M9UX97 Mycobacterium bovis BCG str. Korea 1168P K60_033810 M1IQ04 Mycobacterium liflandii (strain 128FXT) alkB MULP_01451 L7V4G7 Mycobacterium tuberculosis (strain CDC 1551/ alkB MT3350 L7N540 Oshkosh) Mycobacterium canettii CIPT 140070017 alkB BN45_60281 L0QZH1 Mycobacterium canettii CIPT 140070008 alkB BN43_60261 L0QC77 Mycobacterium canettii CIPT 140060008 alkB BN44_70036 L0Q026 Mycobacterium tuberculosis 7199-99 MT7199_3294 L0NZI4 Mycobacterium tuberculosis KZN 605 TBXG_003280 I6RJV1 Mycobacterium tuberculosis KZN 4207 TBSG_03323 I1SDS8 Mycobacterium tuberculosis RGTB327 MRGA327_20020 H8HLB9 Mycobacterium tuberculosis (strain ATCC 35801/TMC alkB ERDMAN_3566 H8EY95 107/Erdman) Mycobacterium tuberculosis UT205 alkB UDA_3252c H6S7Q5 Mycobacterium bovis BCG str. Mexico alkB BCGMEX_3279c G7QY42 Mycobacterium tuberculosis CTRI-2 alkB MTCTRI2_3319 G2N7Q9 Mycobacterium canettii (strain CIPT 140010059) alkB MCAN_32711 G0THM9 Mycobacterium canettii (strain CIPT 140010059) alkB MCAN_32711 G0THM9 Mycobacterium africanum (strain GM041182) alkB MAF_32630 F8M6G6 Mycobacterium tuberculosis (strain CCDC5180) alkB CCDC5180_2963 F7WQM1 CFBR_3446 Mycobacterium tuberculosis (strain CCDC5079) alkB CCDC5079_3000 F7WLN9 CFBS_3441 Mycobacterium tuberculosis (strain KZN 1435/MDR) TBMG_03300 C6DXJ8 Mycobacterium bovis (strain BCG/Tokyo 172/ATCC alkB JTY_3277 C1AH26 35737/TMC 1019) Mycobacterium marinum (strain ATCC BAA-535/M) alkB MMAR_1291 B2HEP2 Mycobacterium tuberculosis (strain F11) TBFG_13281 A5WSG7 Mycobacterium tuberculosis (strain ATCC 25177/ alkB MRA_3293 A5U7S6 H37Ra) Mycobacterium tuberculosis str. Haarlem TBHG_03188 A4KLE9 Mycobacterium bovis (strain BCG/Pasteur 1173P2) alkB BCG_3281c A1KNQ4 Mycobacterium bovis 04-303 O216_17560 V2W1E0 Mycobacterium bovis AN5 O217_17270 V2VQT4 Mycobacterium tuberculosis GuangZ0019 alkB GuangZ0019_1145 T5HDB1 Mycobacterium tuberculosis FJ05194 alkB FJ05194_2026 T5H4I2 Mycobacterium tuberculosis ‘98-R604 INH-RIF-EM’ TBKG_02259 T0EL87 Mycobacterium marinum str. Europe MMEU_4939 S7S303 Mycobacterium marinum MB2 MMMB2_4134 S7QZY6 Mycobacterium orygis 112400015 MORY_17288 M8DBT2 Mycobacterium tuberculosis NCGM2209 alkB NCGM2209_3538 G2UTS8 Mycobacterium bovis BCG str. Moreau RDJ alkB BCGM_3265c F9UZB9 Mycobacterium tuberculosis W-148 TBPG_00365 F2VCH4 Mycobacterium tuberculosis CDC1551A TMMG_02400 E9ZP16 Mycobacterium tuberculosis SUMu012 TMLG_02024 E2WM40 Mycobacterium tuberculosis SUMu011 TMKG_02511 E2WA16 Mycobacterium tuberculosis SUMu010 TMJG_03436 E2VYW3 Mycobacterium tuberculosis SUMu009 TMIG_02769 E2VMD7 Mycobacterium tuberculosis SUMu006 TMFG_00461 E2UQS7 Mycobacterium tuberculosis SUMu005 TMEG_03649 E2UEQ4 Mycobacterium tuberculosis SUMu004 TMDG_02087 E2U2V2 Mycobacterium tuberculosis SUMu003 TMCG_01675 E2TRB4 Mycobacterium tuberculosis SUMu002 TMBG_01947 E2TG69 Mycobacterium tuberculosis SUMu001 TMAG_02705 E1HE07 Mycobacterium africanum K85 TBOG_03815 D6FRF3 Mycobacterium tuberculosis CPHL_A TBNG_02887 D6FLF8 Mycobacterium tuberculosis T46 TBLG_03890 D6F9Q1 Mycobacterium tuberculosis T17 TBJG_02010 D5ZLD1 Mycobacterium tuberculosis GM 1503 TBIG_02964 D5Z897 Mycobacterium tuberculosis 02_1987 TBBG_01719 D5YWK4 Mycobacterium tuberculosis EAS054 TBGG_02463 D5YJM0 Mycobacterium tuberculosis T85 TBEG_02389 D5Y8I4 Mycobacterium tuberculosis T92 TBDG_02114 D5XYS2 Mycobacterium tuberculosis C TBCG_03191 A2VP49 Rhodococcus sp. EsD8 EBESD8_35530 N1M6K3 Amycolatopsis orientalis HCCB10007 AORI_4274 R4SU00 Mycobacterium tuberculosis SUMu008 TMHG_02473 E2VD73 Mycobacterium tuberculosis SUMu007 TMGG_02800 E2V1Z1 Mycobacterium tuberculosis 94_M4241A TBAG_02148 D7EUC2 Gordonia amarae NBRC 15530 alkB GOAMR_02_00080 G7GIN7 Rhodococcus rhodochrous ATCC 21198 RR21198_2302 W4A7D8 Amycolatopsis decaplanina DSM 44594 H074_07696 M2XNH0 Mycobacterium sp. 012931 MMSP_4721 S7R3L1 Rhodococcus erythropolis (strain PR4/NBRC 100887) alkB RER_07460 C0ZPX6 Rhodococcus sp. (strain Q15) alkB1 Q93DN3 Rhodococcus erythropolis CCM2595 O5Y_03630 T1VI31 Rhodococcus sp. P27 N806_28900 U0EPX3 Rhodococcus erythropolis (Arthrobacter picolinophilus) alkB1 Q9XAU0 Rhodococcus qingshengii BKS 20-40 G418_23516 M2V230 Rhodococcus erythropolis SK121 RHOER0001_0742 C3JUT8 Rhodococcus erythropolis DN1 N601_07180 T5HYU5 Nocardia farcinica (strain IFM 10152) NFA_46180 Q5YQS2 Rhodococcus equi NBRC 101255 = C 7 H849_17115 U5DRE7 Shewanella sp. NJ49 alkB1 E3VRS8 Mycobacterium canettii CIPT 140070010 alkB BN42_41302 L0QPN9 Nocardia nova SH22a NONO_c63220 W5TPB1 Rhodococcus equi (strain 103S) (Corynebacterium equi) alkB REQ_33430 E4WK80 Gordonia terrae C-6 GTC6_09699 R7YA99 Nocardioides sp. (strain BAA-499/JS614) Noca_0122 A1SCY2 Gordonia sp. TF6 alkB2 Q5WA49 Hydrocarboniphaga effusa AP103 WQQ_18760 I7ZII6 Gordonia terrae NBRC 100016 alkB GOTRE_037_00320 H5UBE8 Nocardia brasiliensis ATCC 700358 O3I_035145 K0FBU4 Amycolatopsis mediterranei RB B737_6308 T1V895 Amycolatopsis mediterranei (strain S699) (Nocardia AMES_6308 RAM_32810 G0FN68 mediterranei) Amycolatopsis mediterranei (strain U-32) AMED_6400 D8HXC8 Rhodococcus sp. p52 alkB U5S065 Rhodococcus pyridinivorans AK37 AK37_01067 H0JKW2 Rhodococcus pyridinivorans SB3094 Y013_07620 V9XAS5 Janibacter sp. HTCC2649 JNB_17248 A3TPZ2 Gordonia sp. KTR9 KTR9_2914 J9SIP3 Aeromicrobium marinum DSM 15272 HMPREF0063_10220 E2S863 Dietzia cinnamea P4 ES5_02159 E6J5E4 Micromonospora aurantiaca (strain ATCC 27029/DSM Micau_3940 D9T1D7 43813/JCM 10878/NBRC 16125/INA 9442) Dietzia sp. E1 alkB/rub fusion C0LMW6 Rhodococcus ruber BKS 20-38 G352_24171 M2YYB5 Mycobacterium gilvum (strain PYR-GCK) Mflv_3369 A4TAB7 (Mycobacterium flavescens (strain ATCC 700033/PYR-GCK)) Nocardioidaceae bacterium Broad-1 NBCG_03866 E9UYJ8 Rhodococcus rhodochrous ATCC 21198 RR21198_2485 W4A610 Salinisphaera shabanensis E1L3A SSPSH_001855 U2E637 Rhodococcus erythropolis (strain PR4/NBRC 100887) alkB RER_54580 C0ZSH4 Corynebacterium falsenii DSM 44353 CFAL_02965 W5WPK1 Rhodococcus erythropolis CCM2595 O5Y_25995 T1VVR3 gamma proteobacterium BDW918 DOK_04793 I2JMI2 Rhodococcus sp. P27 N806_02390 U0DZR9 Rhodococcus erythropolis DN1 N601_00885 T5IAL6 Rhodococcus erythropolis SK121 RHOER0001_2104 C3JNE0 Rhodococcus qingshengii BKS 20-40 G418_13569 M2WBK9

In some embodiments, the invention provides methods for synthesizing olefinic alcohol products as described above, wherein the enzyme is a long-chain alkane hydroxylase. In some embodiments, the long-chain alkane hydroxylase is selected from Table 4 or a variant thereof having at least 90% identity thereto.

TABLE 4 Long chain alkane hydroxylase enzymes capable of catalyzing selective terminal alkene hydroxylation. Species Origin Gene names Accession No Geobacillus thermodenitrificans (strain NG80-2) ladA GTNG_3499 A4IU28 Geobacillus stearothermophilus (Bacillus A8DC15 stearothermophilus) Paenibacillus sp. JCM 10914 JCM10914_4324 V9GEW8 Bacillus methanolicus MGA3 MGA3_06970 I3E8X7 Geobacillus sp. (strain Y4.1MC1) GY4MC1_0235 E3IA76 Geobacillus thermoglucosidans TNO-09.020 GT20_0226 I0U377 Geobacillus thermoglucosidasius (strain C56-YS93) Geoth_0249 F8CWH6 Bacillus methanolicus PB1 PB1_11994 I3DVL0 Alicyclobacillus acidoterrestris ATCC 49025 N007_16655 T0BMR6 Bhargavaea cecembensis DSE10 ntaA_1 C772_00943 M7P9Z6 Bacillus sp. 1NLA3E B1NLA3E_02955 N0AV94 Burkholderia graminis C4D1M BgramDRAFT_6080 B1G9N8 Burkholderia thailandensis H0587 BTL_4503 W6BDT9 Planomicrobium glaciei CHR43 G159_18855 W3A818 Burkholderia thailandensis E444 BTJ_3656 W6C8D0 Burkholderia thailandensis 2002721723 BTQ_5029 W6BNA4 Burkholderia pseudomallei (strain K96243) BPSS0686 Q63MH2 Burkholderia mallei (strain ATCC 23344) BMAA1146 Q62BX8 Burkholderia thailandensis (strain E264/ATCC 700388/ BTH_II1741 Q2T4G4 DSM 13276/CIP 106301) Burkholderia pseudomallei BPC006 BPC006_II0968 K7QBE8 Burkholderia pseudomallei 1106b BURPS1106B_1056 C5ZS78 Burkholderia pseudomallei MSHR346 GBP346_B0209 C4I1H5 Burkholderia pseudomallei (strain 1106a) BURPS1106A_A0931 A3P3Q7 Burkholderia mallei (strain NCTC 10247) BMA10247_A1520 A3MEL8 Burkholderia mallei (strain NCTC 10229) BMA10229_0093 A2RW50 Burkholderia pseudomallei MSHR338 M218_32405 W1LX20 Burkholderia mallei PRL-20 BMAPRL20_0872 C5N9G7 Burkholderia mallei GB8 horse 4 BMAGB8_A1284 C4B2F1 Burkholderia pseudomallei Pakistan 9 BUH_5241 C0Y1N2 Burkholderia pseudomallei 576 BUC_5105 B7CGH3 Burkholderia pseudomallei S13 BURPSS13_T0065 B1H503 Burkholderia mallei ATCC 10399 BMA10399_L0048 A9LC22 Burkholderia pseudomallei Pasteur 52237 BURPSPAST_J0304 A8KQQ8 Burkholderia pseudomallei 406e BURPS406E_G0092 A8EKE5 Burkholderia mallei JHU BMAJHU_I0303 A5XK99 Burkholderia mallei 2002721280 BMA721280_L0585 A5TFQ0 Alicyclobacillus acidoterrestris ATCC 49025 N007_09450 T0BMI0 Burkholderia pseudomallei MSHR305 BDL_3916 S5NPF6 Burkholderia pseudomallei MSHR146 BBN_4086 W0Q1C4 Burkholderia pseudomallei MSHR511 BBQ_5508 W0MJC7 Burkholderia pseudomallei NAU20B-16 BBS_5466 V9YUR9 Burkholderia pseudomallei NCTC 13178 BBJ_4354 V9YFT0 Burkholderia pseudomallei NCTC 13179 BBK_3804 U5V4E4 Burkholderia pseudomallei MSHR1043 D512_19607 M7EBY4 Burkholderia pseudomallei 1655 BURPS1655_I0183 B2H6F2 Burkholderia pseudomallei 305 BURPS305_5546 A4LI20 Segniliparus rugosus ATCC BAA-974 HMPREF9336_02889 E5XTR7 Burkholderia pseudomallei 1026b BP1026B_II0759 I1WRX2 Burkholderia pseudomallei 354a BP354A_4019 I2MG65 Burkholderia pseudomallei 354e BP354E_3240 I2M2Q7 Burkholderia pseudomallei 1026a BP1026A_2436 I2L127 Burkholderia pseudomallei 1258b BP1258B_3899 I2KY00 Burkholderia pseudomallei 1258a BP1258A_3523 I2KWT0 Pseudomonas putida (strain DOT-T1E) T1E_2746 I7B0Q5 Pseudomonas putida ND6 YSA_09788 I3V2W3 Pseudomonas putida TRO1 C206_18269 N9VYA0 Pseudomonas putida LS46 PPUTLS46_018911 M7RI48 Burkholderia graminis C4D1M BgramDRAFT_6182 B1G9Y6 Burkholderia phytofirmans (strain DSM 17436/PsJN) Bphyt_4538 B2TDZ4 Bhargavaea cecembensis DSE10 moxC_3 C772_02411 M7NEH3 Burkholderia thailandensis MSMB121 BTI_5494 N0APC1 Burkholderia pseudomallei (strain 668) BURPS668_A1016 A3NI44 Burkholderia pseudomallei (strain 1710b) BURPS1710b_A2257 Q3JG95 Burkholderia pseudomallei 1710a BURPS1710A_A0072 C6U1I8 Planomicrobium glaciei CHR43 G159_14295 W3AA87 Burkholderia thailandensis MSMB43 A33K_16732 I6AD68 Pseudomonas sp. GM50 PMI30_04278 J3GFD6 Pseudomonas fluorescens BBc6R8 MHB_001910 V7EA47 Pseudomonas sp. Ag1 A462_06954 J0YEG1 Pseudomonas sp. GM102 PMI18_00569 J2VSE5 Pseudomonas fluorescens (strain SBW25) PFLU_3858 C3JYC1 Pseudomonas sp. (strain M1) PM1_0212365 W5IVB1 Pseudomonas sp. TKP U771_20325 V9R055 Pseudomonas putida (strain F1/ATCC 700007) Pput_3007 A5W4S5 Pseudomonas putida (strain GB-1) PputGB1_1120 B0KS73 Azotobacter vinelandii CA6 seuA AvCA6_43810 M9YDA5 Azotobacter vinelandii CA seuA AvCA_43810 M9Y6B1 Azotobacter vinelandii (strain DJ/ATCC BAA-1303) seuA Avin_43810 C1DGK6 Pseudomonas brassicacearum (strain NFM421) PSEBR_a2282 F2KFH4 Pseudomonas fluorescens Q8r1-96 PflQ8_2313 I4KKG5 Klebsiella oxytoca E718 A225_4709 I6X485 Pseudomonas putida (strain KT2440) PP_2746 Q88JA3 Pseudomonas fluorescens BBc6R8 MHB_002244 V7E7E4 Pseudomonas fluorescens Q2-87 PflQ2_2259 J2EFB8 Pseudomonas sp. Ag1 A462_04671 J0PSS9 Klebsiella oxytoca MGH 42 L388_04093 V3KYZ2 Klebsiella oxytoca 10-5245 HMPREF9689_03721 H3M9T3 Klebsiella oxytoca 10-5243 HMPREF9687_03258 H3LSS6 Klebsiella oxytoca (strain ATCC 8724/DSM 4798/ KOX_01240 G8WD25 JCM 20051/NBRC 3318/NRRL B-199/KCTC 1686) Streptomyces himastatinicus ATCC 53653 SSOG_01846 D9WSJ3 Klebsiella oxytoca MGH 28 L374_04760 V3PRZ9 Klebsiella oxytoca 10-5250 HMPREF9694_02187 H3N1Z4 Klebsiella sp. OBRC7 HMPREF1144_4230 J8VYP0 Klebsiella oxytoca 10-5242 HMPREF9686_03185 H3LCA0 Pantoea ananatis LMG 5342 soxA PANA5342_1855 G9ARF4 Pantoea ananatis PA13 PAGR_g1792 G7UD55 Pantoea ananatis (strain AJ13355) soxA PAJ_1557 F2EW92 Pantoea ananatis (strain LMG 20103) soxA PANA_2246 D4GGW6 Pantoea ananatis BRT175 L585_00145 U4W7P0 Segniliparus rotundus (strain ATCC BAA-972/CDC Srot_2598 D6ZC64 1076/CIP 108378/DSM 44985/JCM 13578) Pantoea stewartii subsp. stewartii DC283 CKS_1871 H3RFH9 Pantoea stewartii subsp. stewartii DC283 CKS_1871 H3RFH9 Rhodococcus opacus M213 WSS_A14179 K8XV73 Klebsiella pneumoniae DMC0799 H217_2899 S7AJY1 Klebsiella pneumoniae 700603 KP700603_18582 M7P910 Klebsiella sp. MS 92-3 HMPREF9538_02211 F3Q553 Klebsiella pneumoniae CG43 D364_16040 U5MF64 Klebsiella pneumoniae subsp. pneumoniae 1084 A79E_0950 K4HBM3 Klebsiella pneumoniae subsp. pneumoniae (strain KPHS_42240 G8VT84 HS11286) Klebsiella pneumoniae KCTC 2242 KPN2242_18760 G0GTG2 Klebsiella pneumoniae NB60 X657_3893 W7K535 Klebsiella pneumoniae EGD-HP19-C N035_09715 W1LTN4 Escherichia coli ISC56 W1HC22 Klebsiella pneumoniae IS33 W1CX87 Klebsiella pneumoniae subsp. pneumoniae BJ1-GA KPST380_90022 W0YH64 Klebsiella pneumoniae subsp. pneumoniae SA1 KPST86_100232 W0XPM0 Klebsiella pneumoniae subsp. pneumoniae T69 SB4536_310004 W0XIP7 Klebsiella pneumoniae MGH 18 L364_01157 V3UHS5 Klebsiella pneumoniae MGH 17 L363_03338 V3SPP7 Klebsiella pneumoniae MGH 21 L367_03372 V3RJW0 Klebsiella pneumoniae MGH 19 L365_03262 V3RDD9 Klebsiella pneumoniae MGH 32 L378_01018 V3P5E6 Klebsiella pneumoniae MGH 30 L376_01094 V3NNG8 Klebsiella pneumoniae MGH 40 L386_03550 V3MB44 Klebsiella pneumoniae MGH 36 L382_03249 V3M7N6 Klebsiella pneumoniae BWH 28 L399_01071 V3JYS4 Klebsiella pneumoniae BWH 30 L401_03358 V3IHX3 Klebsiella pneumoniae UCICRE 2 L413_01241 V3H9M1 Klebsiella pneumoniae UCICRE 7 L418_00976 V3FW02 Klebsiella pneumoniae UCICRE 6 L417_03180 V3FI89 Klebsiella pneumoniae BIDMC 21 L457_03247 V3DWM2 Klebsiella pneumoniae BIDMC 22 L458_03227 V3DGZ8 Klebsiella pneumoniae BIDMC 24 L460_03188 V3BDU6 Klebsiella pneumoniae BIDMC 25 L461_03214 V3B499 Klebsiella pneumoniae BIDMC 40 L477_03188 V3A962 Klebsiella pneumoniae BIDMC 36 L473_03258 V3A6I8 Klebsiella pneumoniae BIDMC 41 L478_00374 V2Z7W2 Klebsiella pneumoniae BIDMC 12C L441_03468 U7BFN1 Klebsiella pneumoniae BIDMC 18C L450_03424 U7AVL5 Klebsiella pneumoniae BIDMC 16 L445_03710 U7AGB1 Enterococcus gallinarum EGD-AAK12 N036_14515 U1CX13 Klebsiella pneumoniae subsp. pneumoniae MP14 KKPNMP14_39700 S8A752 Klebsiella pneumoniae subsp. pneumoniae UKKV901664_39580 S7YC36 UKKV901664 Klebsiella pneumoniae 120_1020 J048_0227 S7I734 Klebsiella pneumoniae 140_1040 J046_0551 S7HZ61 Klebsiella pneumoniae 280_1220 J049_0615 S7H6G5 Klebsiella pneumoniae 160_1080 J047_06104 S7FI27 Klebsiella pneumoniae UHKPC06 H228_0695 S7F6A3 Klebsiella pneumoniae UHKPC67 H212_0084 S7EIH5 Klebsiella pneumoniae UHKPC02 H229_0083 S7EFH7 Klebsiella pneumoniae UHKPC17 H225_0083 S7E3F9 Klebsiella pneumoniae UHKPC31 H227_0223 S7E0F6 Klebsiella pneumoniae UHKPC59 H223_2084 S7DJY5 Klebsiella pneumoniae UHKPC18 H226_0627 S7CZN2 Klebsiella pneumoniae UHKPC61 H220_0228 S7CKP4 Klebsiella pneumoniae UHKPC07 H224_0554 S7C1T8 Klebsiella pneumoniae DMC1316 H219_1515 S7C0U0 Klebsiella pneumoniae UHKPC33 H222_0227 S7BH54 Klebsiella pneumoniae DMC1097 H218_2245 S7A1J0 Klebsiella pneumoniae UHKPC96 H215_0710 S6YYA8 Klebsiella pneumoniae UHKPC77 H214_0083 S6YU31 Klebsiella pneumoniae UHKPC28 H209_0679 S6YQS7 Klebsiella pneumoniae UHKPC69 H213_0083 S6YBZ0 Klebsiella pneumoniae UHKPC47 H211_0128 S6XBP3 Klebsiella pneumoniae UHKPC32 H242_0078 S2J6Y7 Klebsiella pneumoniae UHKPC48 H221_0076 S2I2J3 Klebsiella pneumoniae DMC0526 H216_2445 S2I0S2 Klebsiella pneumoniae VAKPC278 H247_0907 S2H7F7 Klebsiella pneumoniae UHKPC29 H241_0227 S2GQ63 Klebsiella pneumoniae UHKPC05 H210_0554 S2G118 Klebsiella pneumoniae UHKPC45 H239_0077 S2FVN7 Klebsiella pneumoniae UHKPC 52 H234_0218 S2FQ55 Klebsiella pneumoniae 646_1568 J054_0227 S2E5R5 Klebsiella pneumoniae 540_1460 J053_0083 S2E2M9 Klebsiella pneumoniae 440_1540 J051_2140 S2CWI6 Klebsiella pneumoniae 500_1420 J052_0542 S2CKG8 Klebsiella pneumoniae VAKPC309 H252_1202 S2C6A5 Klebsiella pneumoniae KP-11 H254_0775 S2BTB1 Klebsiella pneumoniae 361_1301 J050_2658 S2B565 Klebsiella pneumoniae VAKPC297 H251_0083 S2ACA5 Klebsiella pneumoniae VAKPC270 H249_0897 S1ZBB5 Klebsiella pneumoniae VAKPC280 H248_0984 S1Z9L1 Klebsiella pneumoniae VAKPC276 H250_1158 S1Z4C6 Klebsiella pneumoniae VAKPC269 H246_1198 S1YJN2 Klebsiella pneumoniae VAKPC254 H245_0083 S1XZP2 Klebsiella pneumoniae UHKPC22 H240_0083 S1XYX9 Klebsiella pneumoniae UHKPC04 H243_0549 S1X5H6 Klebsiella pneumoniae VAKPC252 H244_3523 S1WWW4 Klebsiella pneumoniae UHKPC26 H236_0227 S1W5H8 Klebsiella pneumoniae UHKPC27 H233_0552 S1VUY3 Klebsiella pneumoniae UHKPC24 H235_0228 S1V9Y4 Klebsiella pneumoniae UHKPC01 H231_1154 S1V1B9 Klebsiella pneumoniae UHKPC81 H232_2378 S1TWU9 Klebsiella pneumoniae UHKPC40 H207_0083 S1TR15 Klebsiella pneumoniae UHKPC09 H230_0227 S1TQU1 Klebsiella pneumoniae KP-7 H253_1042 S1T453 Klebsiella pneumoniae UHKPC23 H208_0755 R9BIA6 Klebsiella pneumoniae subsp. pneumoniae KpMDU1 C210_21528 N9SXP2 Klebsiella pneumoniae ATCC BAA-1705 KPBAA1705_02256 M7QWX8 Klebsiella pneumoniae ATCC BAA-2146 G000_17665 Kpn2146_4394 M7PZV3 Klebsiella pneumoniae VA360 MTE2 213 M5T2W9 Klebsiella pneumoniae RYC492 KPRYC492_05065 M5Q5H7 Klebsiella pneumoniae RYC492 KPRYC492_05065 M5Q5H7 Klebsiella pneumoniae subsp. pneumoniae KpQ3 B819_29014 M5GIZ6 Klebsiella pneumoniae subsp. pneumoniae Ecl8 BN373_37921 K4UK89 Klebsiella pneumoniae subsp. pneumoniae WGLW5 HMPREF1308_03340 K1NXD5 Klebsiella pneumoniae subsp. pneumoniae WGLW3 HMPREF1307_01233 K1NCK1 Klebsiella pneumoniae subsp. pneumoniae WGLW1 HMPREF1305_01058 K1MMN7 Klebsiella pneumoniae subsp. pneumoniae KPNIH23 KPNIH23_01714 J2W4N5 Klebsiella pneumoniae subsp. pneumoniae KPNIH21 KPNIH21_18909 J2UUP0 Klebsiella pneumoniae subsp. pneumoniae KPNIH18 KPNIH18_04648 J2TP42 Klebsiella pneumoniae subsp. pneumoniae KPNIH17 KPNIH17_07852 J2SZ94 Klebsiella pneumoniae subsp. pneumoniae KPNIH9 KPNIH9_07912 J2PY88 Klebsiella pneumoniae subsp. pneumoniae KPNIH6 KPNIH6_12977 J2NIU0 Klebsiella pneumoniae subsp. pneumoniae KPNIH1 KPNIH1_04615 J2MHH3 Klebsiella pneumoniae subsp. pneumoniae KPNIH22 KPNIH22_01396 J2KA06 Klebsiella pneumoniae subsp. pneumoniae KPNIH19 KPNIH19_02887 J2JA47 Klebsiella pneumoniae subsp. pneumoniae KPNIH16 KPNIH16_07898 J2HIQ1 Klebsiella pneumoniae subsp. pneumoniae KPNIH14 KPNIH14_01932 J2GTK1 Klebsiella pneumoniae subsp. pneumoniae KPNIH11 KPNIH11_05794 J2G1J7 Klebsiella pneumoniae subsp. pneumoniae KPNIH2 KPNIH2_14379 J2BUC4 Klebsiella pneumoniae subsp. pneumoniae KPNIH20 KPNIH20_08348 J2BFJ4 Klebsiella pneumoniae subsp. pneumoniae KPNIH12 KPNIH12_01874 J1YXJ0 Klebsiella pneumoniae subsp. pneumoniae KPNIH10 KPNIH10_07382 J1X9E8 Klebsiella pneumoniae subsp. pneumoniae KPNIH8 KPNIH8_09376 J1WTX7 Klebsiella pneumoniae subsp. pneumoniae KPNIH7 KPNIH7_03054 J1WDZ3 Klebsiella pneumoniae subsp. pneumoniae KPNIH5 KPNIH5_11286 J1V7M9 Klebsiella pneumoniae subsp. pneumoniae KPNIH4 KPNIH4_01334 J1UFY7 Klebsiella sp. 4_1_44FAA HMPREF1024_02306 G9REB7 Klebsiella pneumoniae JM45 N559_1083 S5YDY6 Klebsiella pneumoniae subsp. pneumoniae Kp13 KP13_02362 V9ZFM9 Klebsiella pneumoniae subsp. rhinoscleromatis ATCC HMPREF0484_1763 C8T2C2 13884 Klebsiella pneumoniae subsp. pneumoniae ST258-K26BO BN426_1797 K4RX40 Klebsiella variicola (strain At-22) Kvar_0908 D3RIP8 Klebsiella pneumoniae (strain 342) KPK_0975 B5XUZ5 Klebsiella pneumoniae MGH 20 L366_04030 V3R3V0 Klebsiella pneumoniae UCICRE 10 L421_04096 V3DSZ3 Klebsiella sp. KTE92 A1WC_04002 R8X357 Klebsiella pneumoniae hvKP1 G057_03698 M2A8M6 Mycobacterium hassiacum DSM 44199 C731_0966 K5B980 Klebsiella pneumoniae MGH 48 L394_03318 V3J564 Pantoea vagans (strain C9-1) (Pantoea agglomerans Pvag_pPag10056 E1PKF9 (strain C9-1)) Klebsiella pneumoniae IS22 W1BJB8 Klebsiella pneumoniae subsp. pneumoniae NTUH-K2044 KP1_4424 C4XCS7 Burkholderia sp. CCGE1001 BC1001_4137 E8YTA8 Microvirga lotononidis MicloDRAFT_00046760 I4YVV6 Burkholderia phenoliruptrix BR3459a BUPH_00719 K0DVZ1 Pseudomonas cichorii JBC1 PCH70_03420 W0H3V5 Burkholderia sp. (strain CCGE1003) BC1003_5279 E1TDZ6 Pseudomonas protegens CHA0 soxA1 PFLCHA0_c02440 R4QZ42 Herbaspirillum sp. CF444 PMI16_04881 J2L7C4 Pseudomonas fluorescens (strain Pf-5/ATCC BAA-477) PFL_0243 Q4KK44 Bacillus megaterium WSH-002 BMWSH_4371 G2RTT4 Pseudomonas sp. GM30 PMI25_001642 W6W1D9 Pseudomonas sp. GM78 PMI35_05139 J3D9L9 Pseudomonas sp. GM60 PMI32_02771 J2U6I1 Pseudomonas sp. FH1 H096_21398 W2DLN3 Pseudomonas sp. GM41(2012) PMI27_000125 W6VAV2 Pseudomonas sp. GM67 PMI33_04861 J2TPB1 Pseudomonas fluorescens EGD-AQ6 O204_08695 U1U9U7 Pseudomonas sp. CF161 CF161_31485 S6JVW1 Pseudomonas fluorescens BRIP34879 A986_05371 L7HKJ7 Pseudomonas sp. Lz4W B195_18896 M5QDB7 Collimonas fungivorans (strain Ter331) CFU_2748 G0A9F2 Pseudomonas poae RE*1-1-14 H045_11420 M4K052 Pseudomonas fluorescens BBc6R8 MHB_005864 V7DXC0 Pseudomonas sp. Lz4W B195_14957 M5QFC1 Pseudomonas sp. GM24 PMI23_03232 J2QII9 Pseudomonas sp. GM16 PMI19_05169 J2MFI6 Rhizobium sp. CF080 PMI07_000401 W6W3M6 Pseudomonas sp. FH1 H096_13584 W2DVJ0 Pseudomonas sp. GM25 PMI24_00141 J2Q9Q8 Rhizobium leguminosarum bv. trifolii (strain WSM2304) Rleg2_6510 B6A4D5 Pseudomonas sp. G5(2012) PG5_63250 S2FDS8 Pseudomonas chlororaphis O6 PchlO6_2640 I4XU61 Pseudomonas protegens CHA0 soxA3 PFLCHA0_c26840 R4R5M1 Pseudomonas fluorescens (strain Pf-5/ATCC BAA-477) PFL_2617 Q4KDF9 Rhizobium leguminosarum bv. trifolii WSM597 Rleg9DRAFT_0832 I9N5T8 Bacillus megaterium (strain DSM 319) BMD_1582 D5DC52 Pseudomonas fluorescens WH6 PFWH6_3643 E2XUD7 Rhizobium sp. Pop5 RCCGEPOP_16608 K0VYN0 Bacillus megaterium (strain ATCC 12872/QMB1551) BMQ_0870 D5E197 Pseudomonas cichorii JBC1 PCH70_26220 W0HAE8 Pseudomonas sp. TKP U771_03925 V9QRK7 Pseudomonas aeruginosa C41 Q088_02376 U8DE40 Pseudomonas aeruginosa 62 P997_00130 U9DU73 Pseudomonas aeruginosa BL19 Q073_02117 U8H8T3 Pseudomonas aeruginosa YL84 AI22_19865 W5VAE7 Pseudomonas aeruginosa SCV20265 SCV20265_2995 V9U1K6 Pseudomonas aeruginosa LES431 T223_15220 V9T819 Pseudomonas aeruginosa MTB-1 U769_13585 V5SWN7 Pseudomonas aeruginosa PA1R PA1R_gp0125 U6AQA7 Pseudomonas aeruginosa PA1 PA1S_gp0125 U6A6M8 Pseudomonas aeruginosa PAO1-VE13 N297_2400 U5RPB5 Pseudomonas aeruginosa PAO1-VE2 N296_2400 U5R2L8 Pseudomonas aeruginosa c7447m M802_2397 T2EJL9 Pseudomonas aeruginosa RP73 M062_12135 R9ZF43 Pseudomonas aeruginosa (strain ATCC 15692/PAO1/ PA2326 Q9I1F2 1C/PRS 101/LMG 12228) Pseudomonas aeruginosa (strain UCBPP-PA14) PA14_34540 Q02MC3 Pseudomonas aeruginosa B136-33 G655_13420 M9S636 Pseudomonas aeruginosa DK2 PADK2_13640 I6SJ32 Pseudomonas aeruginosa (strain LESB58) PLES_29781 B7V8Z7 Pseudomonas aeruginosa (strain PA7) PSPA7_2933 A6V5F8 Pseudomonas aeruginosa (strain PA7) PSPA7_2933 A6V5F8 Pseudomonas aeruginosa DHS29 V441_13990 W1QXR3 Pseudomonas aeruginosa MH38 P38_3412 W0WGT3 Pseudomonas aeruginosa VRFPA06 V527_13850 V8HJN2 Pseudomonas aeruginosa VRFPA08 X922_29130 V8DQV8 Pseudomonas aeruginosa DHS01 DPADHS01_13190 V4WR77 Pseudomonas aeruginosa VRFPA01 G039_0203575 V4QMQ4 Pseudomonas aeruginosa HB15 PA15_0330520 V4MN40 Pseudomonas aeruginosa M8A.3 Q082_00075 U9SHI5 Pseudomonas aeruginosa CF27 Q003_00104 U9RU06 Pseudomonas aeruginosa MSH10 Q000_02112 U9RT23 Pseudomonas aeruginosa CF127 Q001_02232 U9RQB8 Pseudomonas aeruginosa CF5 Q004_02036 U9R042 Pseudomonas aeruginosa S54485 Q007_00776 U9QQE4 Pseudomonas aeruginosa BWHPSA007 Q020_00157 U9PK67 Pseudomonas aeruginosa BWHPSA009 Q022_02698 U9NGB4 Pseudomonas aeruginosa BWHPSA008 Q021_00149 U9NF67 Pseudomonas aeruginosa BWHPSA010 Q023_01638 U9MXZ6 Pseudomonas aeruginosa BWHPSA015 Q028_00447 U9MBW2 Pseudomonas aeruginosa BWHPSA016 Q029_01714 U9LQK4 Pseudomonas aeruginosa BL03 Q057_00105 U9LB58 Pseudomonas aeruginosa BL01 Q055_02736 U9KLQ0 Pseudomonas aeruginosa BL02 Q056_06394 U9JUP8 Pseudomonas aeruginosa BL05 Q059_02100 U9JF28 Pseudomonas aeruginosa BL06 Q060_06378 U9IJ92 Pseudomonas aeruginosa BL21 Q075_03038 U9GQQ1 Pseudomonas aeruginosa BL23 Q077_03073 U9FQH5 Pseudomonas aeruginosa BL24 Q078_06288 U9EQY5 Pseudomonas aeruginosa M8A.4 Q083_01720 U9ECA2 Pseudomonas aeruginosa MSH3 P999_02290 U9D2B6 Pseudomonas aeruginosa X24509 Q005_02076 U9CCX5 Pseudomonas aeruginosa UDL Q006_01725 U9C927 Pseudomonas aeruginosa CF18 Q002_02068 U9BVH8 Pseudomonas aeruginosa 19660 Q010_02159 U9AF43 Pseudomonas aeruginosa X13273 Q013_02044 U8Z334 Pseudomonas aeruginosa S35004 Q012_06204 U8YF61 Pseudomonas aeruginosa BWHPSA001 Q014_02765 U8YAB2 Pseudomonas aeruginosa BWHPSA003 Q016_02194 U8XR83 Pseudomonas aeruginosa BWHPSA002 Q015_02292 U8XP62 Pseudomonas aeruginosa BWHPSA004 Q017_02030 U8X7A0 Pseudomonas aeruginosa BWHPSA005 Q018_03069 U8W6E8 Pseudomonas aeruginosa BWHPSA011 Q024_01957 U8VA48 Pseudomonas aeruginosa BWHPSA013 Q026_03028 U8URW4 Pseudomonas aeruginosa BWHPSA012 Q025_02769 U8UQP2 Pseudomonas aeruginosa BWHPSA014 Q027_01719 U8TK96 Pseudomonas aeruginosa BWHPSA017 Q030_05589 U8SKH8 Pseudomonas aeruginosa BWHPSA020 Q033_02593 U8S609 Pseudomonas aeruginosa BWHPSA019 Q032_03133 U8RPR9 Pseudomonas aeruginosa BWHPSA022 Q035_01895 U8R8U4 Pseudomonas aeruginosa BWHPSA023 Q036_00320 U8R6B4 Pseudomonas aeruginosa BWHPSA021 Q034_02035 U8R1N4 Pseudomonas aeruginosa BWHPSA025 Q038_01757 U8PR31 Pseudomonas aeruginosa BWHPSA024 Q037_02761 U8PP93 Pseudomonas aeruginosa BWHPSA027 Q040_02049 U8N8N1 Pseudomonas aeruginosa BL07 Q061_01439 U8LYS6 Pseudomonas aeruginosa BL04 Q058_06192 U8LL05 Pseudomonas aeruginosa BL11 Q065_03099 U8K8S5 Pseudomonas aeruginosa BL10 Q064_02801 U8JQ84 Pseudomonas aeruginosa BL15 Q069_01997 U8IMR3 Pseudomonas aeruginosa BL16 Q070_01957 U8IID0 Pseudomonas aeruginosa BL18 Q072_02105 U8H8J8 Pseudomonas aeruginosa M8A.2 Q081_01961 U8FTG3 Pseudomonas aeruginosa M8A.1 Q080_04721 U8FHJ8 Pseudomonas aeruginosa M9A.1 Q084_05530 U8EPH5 Pseudomonas aeruginosa C20 Q085_03119 U8EML6 Pseudomonas aeruginosa C23 Q086_03122 U8EJ68 Pseudomonas aeruginosa C40 Q087_02201 U8DKJ1 Pseudomonas aeruginosa C48 Q089_02700 U8CPW7 Pseudomonas aeruginosa C51 Q090_05806 U8BVH7 Pseudomonas aeruginosa CF77 Q092_01904 U8BA80 Pseudomonas aeruginosa C52 Q091_05688 U8AZD2 Pseudomonas aeruginosa CF614 Q093_06204 U8ACM4 Pseudomonas aeruginosa VRFPA04 P797_30195 U5AHY5 Pseudomonas aeruginosa HB13 PA13_1029315 U1E3A4 Pseudomonas aeruginosa MSH-10 L346_02111 S0IJJ1 Pseudomonas aeruginosa PA14 CIA_02266 S0I9C6 Pseudomonas aeruginosa PAK PAK_02986 S0I695 Pseudomonas sp. P179 HMPREF1224_05539 N2DDM6 Pseudomonas aeruginosa str. Stone 130 HMPREF1223_07114 N2D7D2 Pseudomonas aeruginosa PA21_ST175 H123_24636 M3AW72 Pseudomonas aeruginosa E2 P998_02032 PAE2_2544 K1DHT6 Pseudomonas aeruginosa ATCC 25324 PABE173_3188 K1DD82 Pseudomonas aeruginosa CI27 PACI27_2786 K1CTB3 Pseudomonas aeruginosa ATCC 700888 PABE177_2660 K1CGR7 Pseudomonas aeruginosa ATCC 14886 PABE171_3115 K1BXJ5 Pseudomonas aeruginosa PADK2_CF510 CF510_22344 I1ACS3 Pseudomonas aeruginosa MPAO1/P2 O1Q_15090 H3TFC3 Pseudomonas aeruginosa MPAO1/P1 O1O_28545 H3T6G4 Pseudomonas sp. 2_1_26 HMPREF1030_05556 G5G1F3 Pseudomonas aeruginosa 2192 PA2G_01431 A3LB74 Pseudomonas aeruginosa C3719 PACG_01235 A3KU95 Erwinia billingiae (strain Eb661) EbC_20720 D8MRZ6 Xanthomonas axonopodis pv. citri (strain 306) XAC0855 Q8PP33 Xanthomonas citri subsp. citri Aw12879 XCAW_03724 M4W2T5 Xanthomonas axonopodis Xac29-1 XAC29_04355 M4U7K3 Xanthomonas citri pv. mangiferaeindicae LMG 941 ladA XMIN_2789 H8FHG1 Xanthomonas axonopodis pv. punicae str. LMG 859 ladA XAPC_728 H1XCV7 Leifsonia aquatica ATCC 14665 N136_01626 U2TBF7 Serratia marcescens subsp. marcescens Db11 SMDB11_2421 V6A0D9 Pseudomonas aeruginosa VRFPA05 T266_33830 V4WJP9 Pseudomonas aeruginosa BL22 Q076_01761 U9GCW5 Pseudomonas aeruginosa BL22 Q076_01761 U9GCW5 Xanthomonas axonopodis pv. malvacearum str. MOU_00060 K8GBN4 GSPB1386 Pseudomonas aeruginosa VRFPA07 X778_28580 V8E3G0 Pseudomonas aeruginosa BL20 Q074_02826 U9HSV9 Pseudomonas aeruginosa BL25 Q079_01143 U9F0W8 Pseudomonas aeruginosa BL09 Q063_00187 U8L2Y0 Serratia marcescens WW4 SMWW4_v1c31920 L7ZQQ5 Serratia marcescens VGH107 F518_24469 M3BTM0 Pseudomonas aeruginosa BWHPSA018 Q031_00379 U8TSK3 Pseudomonas aeruginosa M18 PAM18_2715 G2L1H6 Pseudomonas aeruginosa BL12 Q066_03852 U9I855 Pseudomonas aeruginosa BWHPSA028 Q041_02218 U8NES6 Pseudomonas aeruginosa WC55 L683_26830 T5KSU5 Pseudomonas aeruginosa NCMG1179 NCGM1179_2739 G2U5R3 Rhodococcus erythropolis SK121 RHOER0001_2299 C3JDL9 Pseudomonas aeruginosa VRFPA03 M770_16185 W1MK34 Pseudomonas aeruginosa BL13 Q067_03184 U9I925 Serratia marcescens EGD-HP20 N040_11055 U1TLQ0 Pseudomonas aeruginosa NCGM2.S1 NCGM2_3338 G4LI50 Pseudomonas aeruginosa 39016 PA39016_002700003 E3A2U8 Pseudomonas aeruginosa MH27 PAMH27_2887 V6AFD9 Pseudomonas aeruginosa JJ692 Q008_02805 U9PMT7 Pseudomonas aeruginosa 6077 Q011_02150 U9ATK4 Pseudomonas aeruginosa U2504 Q009_02593 U9AAM5 Pseudomonas aeruginosa BWHPSA006 Q019_02936 U8VL16 Pseudomonas aeruginosa BL08 Q062_04340 U8KSZ8 Pseudomonas aeruginosa BL14 Q068_02182 U8JUF2 Pseudomonas aeruginosa BL17 Q071_02971 U8H8J5 Pseudomonas aeruginosa PA45 H734_07342 N4W202 Rhodococcus erythropolis CCM2595 O5Y_21155 T1VSG7 Rhodococcus sp. P27 N806_09240 U0ED84 Kosakonia radicincitans DSM 16656 Y71_0158 J1QW00 Rhodococcus erythropolis (strain PR4/NBRC 100887) RER_45000 C0ZMF0 Klebsiella pneumoniae MGH 46 L392_03264 V3LZ98 Klebsiella pneumoniae MGH 44 L390_02205 V3JUR2 Klebsiella pneumoniae UCICRE 4 L415_03363 V3FXF6 Klebsiella pneumoniae 303K N598_24365 U6T101 Klebsiella pneumoniae UHKPC179 H238_2267 S7F9A7 Klebsiella pneumoniae UHKPC57 H237_2247 S2EDB5 Klebsiella pneumoniae JHCK1 MTE1_213 M3U9Q5 Klebsiella pneumoniae subsp. pneumoniae WGLW2 HMPREF1306_03733 K1NBI6 Klebsiella pneumoniae UCICRE 14 L425_03054 V3CJD9 Rhodococcus qingshengii BKS 20-40 G418_04858 M2XMT9 Pantoea sp. Sc1 S7A_19914 H8DUB8 Klebsiella sp. 1_1_55 HMPREF0485_02899 D6GIG4 Pantoea agglomerans Tx10 L584_13665 U4VW62 Escherichia coli 909957 HMPREF1619_02817 V0B421 Klebsiella pneumoniae KP-1 KLP1_1662 U2ABR1 Rhodococcus erythropolis DN1 N601_05680 T5I9L8 Klebsiella pneumoniae UCICRE 8 L419_03300 V3F3T1 Brenneria sp. EniD312 BrE312_1717 G7LVX2 Klebsiella pneumoniae BIDMC 23 L459_03205 V3BAE8 Raoultella ornithinolytica B6 RORB6_23555 M9W8P0 Klebsiella oxytoca 10-5246 HMPREF9690_03902 H3MRJ7 Pantoea agglomerans 299R F385_1445 L7BV82 Pantoea sp. aB PanABDRAFT_3926 E0M3F8 Pseudomonas sp. CFII64 CFII64_23274 S6GXI3 Pseudomonas synxantha BG33R PseBG33_0275 I4KV50 Pseudomonas syringae pv. actinidiae ICMP 18801 A221_07756 S6XYV3 Pseudomonas syringae pv. actinidiae ICMP 19072 A3SO_07400 S6PNP2 Pseudomonas syringae pv. actinidiae ICMP 19073 A262_20054 S6MLA8 Pseudomonas syringae pv. actinidiae ICMP 19071 A264_07551 S6M2E1 Pseudomonas syringae pv. actinidiae ICMP 19104 A258_19792 S6QSB5 Pseudomonas syringae pv. actinidiae ICMP 9855 A252_19596 S6QRN6 Pseudomonas syringae pv. actinidiae ICMP 19102 A253_19857 S6Q6B9 Pseudomonas syringae pv. actinidiae ICMP 19068 A260_20086 S6Q126 Pseudomonas syringae pv. theae ICMP 3923 A584_21008 S6MKD2 Pseudomonas syringae pv. actinidiae ICMP 19103 A256_19800 S6M4P1 Rhizobium leguminosarum bv. viciae (strain 3841) pRL90300 Q1M8E2 Pseudomonas sp. GM25 PMI24_01694 J2PHH1 Herbaspirillum sp. YR522 PMI40_00700 J3HY53 Pseudomonas syringae pv. morsprunorum str. M302280 PSYMP_05599 F3DS65 Pseudomonas fluorescens (strain Pf0-1) Pfl01_0238 Q3KJS4 Pseudomonas avellanae BPIC 631 Pav631_4731 K2RRZ8 Pseudomonas fluorescens R124 I1A_000262 K0W8U4 Pseudomonas syringae pv. syringae (strain B728a) Psyr_2869 Q4ZSG7 Pseudomonas syringae CC1557 N018_12850 W0MW63 Pseudomonas sp. GM80 PMI37_03766 J3DKC5 Pseudomonas syringae pv. syringae SM PssSM_2902 S3MKC4 Pseudomonas syringae pv. avellanae str. ISPaVe037 Pav037_2494 K2T3F9 Pseudomonas syringae pv. aceris str. M302273 PSYAR_06142 F3JE47 Pseudomonas syringae pv. maculicola str. ES4326 PMA4326_07981 F3HHE2 Pseudomonas syringae BRIP39023 A988_19986 L7GSY0 Pseudomonas syringae pv. aptata str. DSM 50252 PSYAP_18083 F3J2D2 Pseudomonas savastanoi pv. savastanoi NCPPB 3335 PSA3335_0550 D7HUP0 Pseudomonas syringae pv. aesculi str. 0893_23 PSYAE_00125 F3D7S6 Pseudomonas syringae BRIP34881 A987_17762 L7G2P2 Pseudomonas syringae BRIP34876 A979_21556 L7FTL3 Rhizobium leguminosarum bv. viciae WSM1455 Rleg5DRAFT_0033 J0URT9 Pseudomonas syringae Cit 7 PSYCIT7_07619 F3GWQ5 Acinetobacter baumannii NIPH 410 F910_02332 S3TEC4 Acinetobacter baumannii OIFC110 ACIN5110_2029 K5S1X4 Acinetobacter baumannii WC-692 ACINWC692_1619 K1ER91 Pseudomonas sp. TKP U771_01460 V9QPN2 Pseudomonas syringae pv. syringae B64 PssB64_3039 L8NFP3 Pseudomonas syringae pv. actinidiae ICMP 19094 A241_11585 S6VCM5 Pseudomonas syringae pv. actinidiae ICMP 18883 A243_23241 S6TZP7 Pseudomonas syringae pv. actinidiae ICMP 19095 A242_23680 S6TDL4 Pseudomonas syringae pv. actinidiae ICMP 19099 A247_15969 S6S3V9 Pseudomonas syringae pv. actinidiae ICMP 19100 A248_23237 S6R962 Pseudomonas syringae pv. actinidiae ICMP 19098 A246_16023 S6LVQ8

In some embodiments, the invention provides methods for synthesizing olefinic alcohol products as described above, wherein the enzyme is a cytochrome P450. In some embodiments, the cytochrome P450 is selected from Table 5 or a variant thereof having at least 90% identity thereto. In some embodiments, the cytochrome P450 is a member of the CYP52 or CYP153 family.

TABLE 5 Cytochrome P450 enzymes capable of catalyzing selective terminal alkene hydroxylation. Species Origin Gene names Accession No Candida tropicalis (Yeast) CYP52A12 Q874J5 Candida tropicalis (strain ATCC MYA-3404/T1) (Yeast) CTRG_02725 C5M8K3 Candida tropicalis (Yeast) CYP52A6 P30608 Candida albicans (Yeast) Q9C2X5 Candida maltosa (Yeast) CYP52A3-B P24458 Candida dubliniensis (strain CD36/ATCC MYA-646/ CYP52A5 CD36_64140 B9WJ64 CBS 7987/NCPF 3949/NRRL Y-17841) (Yeast) Candida albicans (strain SC5314/ATCC MYA-2876) ALK1 CaO19.13150 Q5A8M1 (Yeast) orf19.13150 Candida albicans (strain SC5314/ATCC MYA-2876) ALK1 CaO19.5728 Q5A8U5 (Yeast) orf19.5728 Candida maltosa (strain Xu316) (Yeast) G210_4862 M3HRI7 Candida maltosa (Yeast) CYP52A3-A P16496 Candida orthopsilosis (strain 90-125) (Yeast) CORT_0F01930 H8X8E5 Candida parapsilosis (strain CDC 317/ATCC MYA-4646) CPAR2_600870 G8B4X9 (Yeast) (Monilia parapsilosis) Lodderomyces elongisporus (strain ATCC 11503/CBS LELG_04957 A5E5R8 2605/JCM 1781/NBRC 1676/NRRL YB-4239) (Yeast) (Saccharomyces elongisporus) Candida maltosa (Yeast) ALK3-B (CYP52A4) B0VX53 Candida maltosa (Yeast) ALK8-B Q12584 Candida tropicalis (Yeast) CYP52A8 P30610 Debaryomyces hansenii (strain ATCC 36239/CBS 767/ DEHA2E18634g Q6BNV8 JCM 1990/NBRC 0083/IGC 2968) (Yeast) (Torulaspora hansenii) Candida tropicalis (Yeast) CYP52A17 Q874I9 Candida maltosa (strain Xu316) (Yeast) G210 3820 M3II00 Spathaspora passalidarum (strain NRRL Y-27907/11-Y1) SPAPADRAFT_59378 G3AJR6 Scheffersomyces stipitis (strain ATCC 58785/CBS 6054/ CP52M PICST_58031 A3LRT5 NBRC 10063/NRRL Y-11545) (Yeast) (Pichia stipitis) Candida parapsilosis (strain CDC 317/ATCC MYA-4646) CPAR2_503950 G8BH23 (Yeast) (Monilia parapsilosis) Candida parapsilosis (strain CDC 317/ATCC MYA-4646) CPAR2_800510 G8BBI4 (Yeast) (Monilia parapsilosis) Candida tropicalis (Yeast) CYP52A18 Q874I8 Candida maltosa (strain Xu316) (Yeast) G210_4812 M3K5V3 Debaryomyces hansenii (Yeast) (Torulaspora hansenii) CYP52A13 ALK2 Q9Y758 Meyerozyma guilliermondii (strain ATCC 6260/CBS 566/ PGUG_05855 A5DRF4 DSM 6381/JCM 1539/NBRC 10279/NRRL Y-324) (Yeast) (Candida guilliermondii) Debaryomyces hansenii (strain ATCC 36239/CBS 767/ DEHA2C02596g Q6BVH7 JCM 1990/NBRC 0083/IGC 2968) (Yeast) (Torulaspora hansenii) Candida maltosa (Yeast) CYP52A5 Q12581 Meyerozyma guilliermondii (strain ATCC 6260/CBS 566/ PGUG_01238 A5DD87 DSM 6381/JCM 1539/NBRC 10279/NRRL Y-324) (Yeast) (Candida guilliermondii) Debaryomyces hansenii (Yeast) (Torulaspora hansenii) CYP52A12 ALK1 Q9Y757 Candida dubliniensis (strain CD36/ATCC MYA-646/ CYP52A14 CD36_25250 B9WKL6 CBS 7987/NCPF 3949/NRRL Y-17841) (Yeast) Meyerozyma guilliermondii (strain ATCC 6260/CBS 566/ PGUG_05670 A5DQW9 DSM 6381/JCM 1539/NBRC 10279/NRRL Y-324) (Yeast) (Candida guilliermondii) Candida albicans (strain SC5314/ATCC MYA-2876) ALK2 CaO19.7513 Q5AAH6 (Yeast) orf19.7513 Candida albicans (strain WO-1) (Yeast) CAWG_01382 C4YNC3 Candida tropicalis (Yeast) CYP52A14 CYP14 Q874J3 Candida tropicalis (Yeast) CYP52A13 Q874J4 Pichia sorbitophila (strain ATCC MYA-4447/BCRC Piso0_002820 G8YG24 22081/CBS 7064/NBRC 10061/NRRL Y-12695) GNLVRS01_PISO0I18532g (Hybrid yeast) Candida parapsilosis (strain CDC 317/ATCC MYA- CPAR2_204220 G8BFZ5 4646) (Yeast) (Monilia parapsilosis) Candida tropicalis (Yeast) CYP52A20 Q874I6 Candida tropicalis (Yeast) CYP52A19 Q874I7 Lodderomyces elongisporus (strain ATCC 11503/CBS LELG_00044 A5DRQ8 2605/JCM 1781/NBRC 1676/NRRL YB-4239) (Yeast) (Saccharomyces elongisporus) Candida albicans (strain WO-1) (Yeast) CAWG_02011 C4YMD2 Candida albicans (strain SC5314/ATCC MYA-2876) ALK8 CaO19.10 Q59K96 (Yeast) CaO19.7683 Candida albicans (Yeast) alk8 O74626 Candida maltosa (strain Xu316) (Yeast) G210_4811 M3JDC1 Scheffersomyces stipitis (strain ATCC 58785/CBS 6054/ CP52C PICST_56580 A3LR60 NBRC 10063/NRRL Y-11545) (Yeast) (Pichia stipitis) Lodderomyces elongisporus (strain ATCC 11503/CBS LELG_03506 A5E1L9 2605/JCM 1781/NBRC 1676/NRRL YB-4239) (Yeast) (Saccharomyces elongisporus) Candida tropicalis (strain ATCC MYA-3404/T1) (Yeast) CTRG_03115 C5MAM3 Pichia sorbitophila (strain ATCC MYA-4447/BCRC Piso0_002820 G8YDL5 22081/CBS 7064/NBRC 10061/NRRL Y-12695) GNLVRS01_PISO0J20293g (Hybrid yeast) Candida parapsilosis (strain CDC 317/ATCC MYA-4646) CPAR2_204210 G8BFZ4 (Yeast) (Monilia parapsilosis) Spathaspora passalidarum (strain NRRL Y-27907/11-Y1) SPAPADRAFT_134963 G3AJD3 Candida tropicalis (strain ATCC MYA-3404/T1) (Yeast) CTRG_01061 C5M4S1 Candida tropicalis (Yeast) CYP52A2 P30607 Candida parapsilosis (strain CDC 317/ATCC MYA-4646) CPAR2_800520 G8BBI5 (Yeast) (Monilia parapsilosis) Scheffersomyces stipitis (strain ATCC 58785/CBS 6054/ CP52L PICST_56638 A3LSP0 NBRC 10063/NRRL Y-11545) (Yeast) (Pichia stipitis) Candida parapsilosis (strain CDC 317/ATCC MYA-4646) CPAR2_203780 G8BFV1 (Yeast) (Monilia parapsilosis) Candida maltosa (strain Xu316) (Yeast) G210_4902 M3IU34 Candida orthopsilosis (strain 90-125) (Yeast) CORT_0D03890 H8X5Y1 Candida dubliniensis (strain CD36/ATCC MYA-646/ CD36_32710 B9WMB3 CBS 7987/NCPF 3949/NRRL Y-17841) (Yeast) Pichia sorbitophila (strain ATCC MYA-4447/BCRC G8YJP0 22081/CBS 7064/NBRC 10061/NRRL Y-12695) (Hybrid yeast) Debaryomyces hansenii (strain ATCC 36239/CBS 767/ DEHA2E18590g Q6BNW0 JCM 1990/NBRC 0083/IGC 2968) (Yeast) (Torulaspora hansenii) Candida maltosa (Yeast) CYP52A9 Q12586 Scheffersomyces stipitis (strain ATCC 58785/CBS 6054/ ALK2 PICST_35590 A3LS01 NBRC 10063/NRRL Y-11545) (Yeast) (Pichia stipitis) Spathaspora passalidarum (strain NRRL Y-27907/11-Y1) SPAPADRAFT_67265 G3APG2 Candida tropicalis (strain ATCC MYA-3404/T1) (Yeast) CTRG_03120 C5MAM8 Candida maltosa (Yeast) CYP52A11 Q12589 Candida albicans (strain WO-1) (Yeast) CAWG_01383 C4YNC4 Candida tropicalis (strain ATCC MYA-3404/T1) (Yeast) CTRG_01060 C5M4S0 Candida albicans (strain SC5314/ATCC MYA-2876) ALK3 CaO19.7512 Q5AAH7 (Yeast) orf19.7512 Candida tropicalis (Yeast) CYP52A1 P10615 Scheffersomyces stipitis (strain ATCC 58785/CBS 6054/ CYP52 PICST_37142 A3LZV9 NBRC 10063/NRRL Y-11545) (Yeast) (Pichia stipitis) Debaryomyces hansenii (strain ATCC 36239/CBS 767/ DEHA2E18612g Q6BNV9 JCM 1990/NBRC 0083/IGC 2968) (Yeast) (Torulaspora hansenii) Candida tenuis (strain ATCC 10573/BCRC 21748/CBS CANTEDRAFT_115474 G3BA51 615/JCM 9827/NBRC 10315/NRRL Y-1498/VKM Y-70) (Yeast) Lodderomyces elongisporus (strain ATCC 11503/CBS LELG_03309 A5E122 2605/JCM 1781/NBRC 1676/NRRL YB-4239) (Yeast) (Saccharomyces elongisporus) Lodderomyces elongisporus (strain ATCC 11503/CBS LELG_03505 A5E1L8 2605/JCM 1781/NBRC 1676/NRRL YB-4239) (Yeast) (Saccharomyces elongisporus) Candida tropicalis (Yeast) CYP52A16 CYP16 Q874J1 Candida tropicalis (Yeast) CYP52A15 Q874J2 Candida maltosa (Yeast) CYP52A10 Q12588 Candida dubliniensis (strain CD36/ATCC MYA-646/ ALK3-A CD36_25260 B9WKL7 CBS 7987/NCPF 3949/NRRL Y-17841) (Yeast) Candida maltosa (Yeast) CYP52A4 P16141 Candida tenuis (strain ATCC 10573/BCRC 21748/CBS CANTEDRAFT_113909 G3B3X3 615/JCM 9827/NBRC 10315/NRRL Y-1498/VKM Y-70) (Yeast) Meyerozyma guilliermondii (Yeast) (Candida CYP52 I6UGD5 guilliermondii) Spathaspora passalidarum (strain NRRL Y-27907/11-Y1) SPAPADRAFT_153278 G3AMY8 Candida tenuis (strain ATCC 10573/BCRC 21748/CBS CANTEDRAFT_116673 G3BEU9 615/JCM 9827/NBRC 10315/NRRL Y-1498/VKM Y-70) (Yeast) Candida maltosa (strain Xu316) (Yeast) G210_3821 M3J257 Candida tropicalis (Yeast) CYP52A7 P30609 Clavispora lusitaniae (strain ATCC 42720) (Yeast) CLUG_03984 C4Y750 (Candida lusitaniae) Debaryomyces hansenii (strain ATCC 36239/CBS 767/ DEHA2C01100g Q6BVP2 JCM 1990/NBRC 0083/IGC 2968) (Yeast) (Torulaspora hansenii) Candida tropicalis (Yeast) CYP52D2 Q874J0 Clavispora lusitaniae (strain ATCC 42720) (Yeast) CLUG_04851 C4Y9G1 (Candida lusitaniae) Meyerozyma guilliermondii (strain ATCC 6260/CBS 566/ PGUG_04005 A5DL54 DSM 6381/JCM 1539/NBRC 10279/NRRL Y-324) (Yeast) (Candida guilliermondii) Yarrowia lipolytica (Candida lipolytica) ALK6 O74132 Yarrowia lipolytica (strain CLIB 122/E 150) (Yeast) YALI0_B01848g F2Z623 (Candida lipolytica) Yarrowia lipolytica (strain CLIB 122/E 150) (Yeast) YALI0_E25982g Q6C4K6 (Candida lipolytica) Yarrowia lipolytica (Candida lipolytica) ALK1 O74127 Yarrowia lipolytica (Candida lipolytica) ALK2 O74128 Yarrowia lipolytica (strain CLIB 122/E 150) (Yeast) YALI0_F01320g F2Z6J3 (Candida lipolytica) Candida maltosa (Yeast) CYP52D1 Q12585 Yarrowia lipolytica (strain CLIB 122/E 150) (Yeast) YALI0_B20702g Q6CDW4 (Candida lipolytica) Byssochlamys spectabilis (strain No. 5/NBRC 109023) PVAR5_4403 V5G4E7 (Paecilomyces variotii) Byssochlamys spectabilis (strain No. 5/NBRC 109023) PVAR5_4403 V5G4E7 (Paecilomyces variotii) Aspergillus terreus (strain NIH 2624/FGSC A1156) ATEG_02198 Q0CVT6 Neosartorya fischeri (strain ATCC 1020/DSM 3700/ NFIA_029600 A1D9P7 FGSC A1164/NRRL 181) (Aspergillus fischerianus) Yarrowia lipolytica (Candida lipolytica) ALK4 O74130 Yarrowia lipolytica (strain CLIB 122/E 150) (Yeast) YALI0_B13816g F2Z6H3 (Candida lipolytica) Penicillium digitatum (strain PHI26/CECT 20796) PDIG_58170 K9G9Y0 (Green mold) Penicillium digitatum (strain Pd1/CECT 20795) (Green PDIP_67660 K9FGZ9 mold) Aspergillus niger (strain ATCC 1015/CBS 113.46/ ASPNIDRAFT_210944 G3XNK4 FGSC A1144/LSHB Ac4/NCTC 3858a/NRRL 328/ USDA 3528.7) Aspergillus niger (strain CBS 513.88/FGSC A1513) An14g01110 A2R2K9 Tuber melanosporum (strain Mel28) (Perigord black GSTUM_00009186001 D5GJT6 truffle) Yarrowia lipolytica (Candida lipolytica) ALK7 O74133 Yarrowia lipolytica (strain CLIB 122/E 150) (Yeast) YALI0_A15488g F2Z6A4 (Candida lipolytica) Arthrobotrys oligospora (strain ATCC 24927/CBS AOL_s00109g132 G1XKA3 115.81/DSM 1491) (Nematode-trapping fungus) (Didymozoophaga oligospora) Dactylellina haptotyla (strain CBS 200.50) (Nematode- H072_6900 S8ADY3 trapping fungus) (Monacrosporium haptotylum) Yarrowia lipolytica (strain CLIB 122/E 150) (Yeast) YALI0_B06248g Q6CFK2 (Candida lipolytica) Aspergillus clavatus (strain ATCC 1007/CBS 513.65/ ACLA_054640 A1C993 DSM 816/NCTC 3887/NRRL 1) Byssochlamys spectabilis (strain No. 5/NBRC 109023) PVAR5_0196 V5FIS1 (Paecilomyces variotii) Aspergillus kawachii (strain NBRC 4308) (White koji AKAW_05280 G7XJE1 mold) (Aspergillus awamori var. kawachi) Aspergillus oryzae (strain 3.042) (Yellow koji mold) Ao3042_00039 I8AC74 Aspergillus flavus (strain ATCC 200026/FGSC A1120/ AFLA_041790 B8NCU4 NRRL 3357/JCM 12722/SRRC 167) Aspergillus oryzae (strain ATCC 42149/RIB 40) (Yellow AO090011000346 Q2U0Q3 koji mold) Aspergillus oryzae (Yellow koji mold) CYP52H3 D4QC14 Candida tenuis (strain ATCC 10573/BCRC 21748/CBS CANTEDRAFT_130130 G3B1J0 615/JCM 9827/NBRC 10315/NRRL Y-1498/VKM Y-70) (Yeast) Emericella nidulans (strain FGSC A4/ATCC 38163/ AN7131.2 ANIA_07131 Q5AX49 CBS 112.46/NRRL 194/M139) (Aspergillus nidulans) Talaromyces stipitatus (strain ATCC 10500/CBS 375.48/ TSTA_012000 B8ME14 QM 6759/NRRL 1006) (Penicillium stipitatum) Starmerella bombicola B8QHP3 Hordeum vulgare var. distichum (Two-rowed barley) F2E8C2 Mycosphaerella graminicola (strain CBS 115943/ CYP-27 F9X9F0 IPO323) (Speckled leaf blotch fungus) (Septoria tritici) MYCGRDRAFT_70822 Neosartorya fumigata (strain ATCC MYA-4609/Af293/ AFUA_4G03800 Q4W9T4 CBS 101355/FGSC A1100) (Aspergillus fumigatus) Neosartorya fumigata (strain CEA10/CBS 144.89/ AFUB_099220 B0YEH7 FGSC A1163) (Aspergillus fumigatus) Penicillium chrysogenum (strain ATCC 28089/DSM Pc14g00320 B6H5K4 1075/Wisconsin 54-1255) (Penicillium notatum) PCH_Pc14g00320 Clavispora lusitaniae (strain ATCC 42720) (Yeast) CLUG_04098 C4Y4W0 (Candida lusitaniae) Penicillium roqueforti CYP52A12PROQFM164_S0 W6QFZ4 3g001613 Yarrowia lipolytica (Candida lipolytica) ALK5 O74131 Yarrowia lipolytica (strain CLIB 122/E 150) (Yeast) YALI0_B13838g F2Z5W7 (Candida lipolytica) Candida tenuis (strain ATCC 10573/BCRC 21748/CBS CANTEDRAFT_107892 G3B8A7 615/JCM 9827/NBRC 10315/NRRL Y-1498/VKM Y-70) (Yeast) Penicillium marneffei (strain ATCC 18224/CBS 334.59/ PMAA_059650 B6QM59 QM 7333) Yarrowia lipolytica (strain CLIB 122/E 150) (Yeast) YALI0_A20130g Q6CGD9 (Candida lipolytica) Candida apicola (Yeast) CYP52E2 Q12573 Macrophomina phaseolina (strain MS6) (Charcoal rot MPH_10814 K2RGW0 fungus) Cyphellophora europaea CBS 101466 HMPREF1541 06043 W2RTP6 Cochliobolus sativus (strain ND90Pr/ATCC 201652) COCSADRAFT_40532 M2STM3 (Common root rot and spot blotch fungus) (Bipolaris sorokiniana) Cochliobolus sativus (strain ND90Pr/ATCC 201652) COCSADRAFT_148934 M2SE93 (Common root rot and spot blotch fungus) (Bipolaris sorokiniana) Bipolaris victoriae FI3 COCVIDRAFT_89176 W7ES92 Bipolaris zeicola 26-R-13 COCCADRAFT_102357 W6Y6Y4 Cochliobolus heterostrophus (strain C5/ATCC 48332/ COCHEDRAFT_1085873 M2TJW9 race O) (Southern corn leaf blight fungus) (Bipolaris maydis) Cochliobolus heterostrophus (strain C4/ATCC 48331/ COCC4DRAFT_201005 N4WTS2 race T) (Southern corn leaf blight fungus) (Bipolaris maydis) Pseudogymnoascus destructans (strain ATCC MYA-4855/ GMDG_04968 L8GCB9 20631-21) (Bat white-nose syndrome fungus) (Geomyces destructans) Aspergillus terreus (strain NIH 2624/FGSC A1156) ATEG_03903 Q0CQY1 Marssonina brunnea f. sp. multigermtubi (strain MB_m1) MBM_06876 K1WCN3 (Marssonina leaf spot fungus) Penicillium marneffei (strain ATCC 18224/CBS 334.59/ PMAA_093890 B6QHD0 QM 7333) Neosartorya fumigata (strain CEA10/CBS 144.89/ AFUB_025410 B0XRZ8 FGSC A1163) (Aspergillus fumigatus) Candida apicola (Yeast) CYP52E1 P43083 Neosartorya fumigata (strain ATCC MYA-4609/Af293/ AFUA_2G09540 Q4X1L5 CBS 101355/FGSC A1100) (Aspergillus fumigatus) Neosartorya fischeri (strain ATCC 1020/DSM 3700/ NFIA_085030 A1DGP3 FGSC A1164/NRRL 181) (Aspergillus fischerianus) Cordyceps militaris (strain CM01) (Caterpillar fungus) CCM_07376 G3JQU8 Coniosporium apollinis (strain CBS 100218) (Rock- W97_02755 R7YNR2 inhabiting black yeast) Penicillium chrysogenum (strain ATCC 28089/DSM Pc22g19240 B6HVQ7 1075/Wisconsin 54-1255) (Penicillium notatum) PCH_Pc22g19240 Penicillium digitatum (strain Pd1/CECT 20795) (Green PDIP_65200 K9G3N2 mold) Penicillium digitatum (strain PHI26/CECT 20796) PDIG_30820 K9FYP6 (Green mold) Penicillium roqueforti PROQFM164_S01g001598 W6QDZ0 Marssonina brunnea f. sp. multigermtubi (strain MB_m1) MBM_06372 K1WQC3 (Marssonina leaf spot fungus) Botryotinia fuckeliana (strain BcDW1) (Noble rot fungus) BcDW1_3993 M7U1E8 (Botrytis cinerea) Botryotinia fuckeliana (strain T4) (Noble rot fungus) BofuT4P27000003001 G2Y6G5 (Botrytis cinerea) Emericella nidulans (strain FGSC A4/ATCC 38163/ AN9384.2 ANIA_09384 Q5AQP6 CBS 112.46/NRRL 194/M139) (Aspergillus nidulans) Candida maltosa (Yeast) CYP52C2 Q12587 Phaeosphaeria nodorum (strain SN15/ATCC MYA-4574/ SNOG_02153 Q0V1G1 FGSC 10173) (Glume blotch fungus) (Septoria nodorum) Pyrenophora tritici-repentis (strain Pt-1C-BFP) (Wheat tan PTRG_08257 B2WF96 spot fungus) (Drechslera tritici-repentis) Pyrenophora teres f. teres (strain 0-1) (Barley net blotch PTT_00451 E3RCI0 fungus) (Drechslera teres f. teres) Aspergillus niger (strain ATCC 1015/CBS 113.46/ ASPNIDRAFT_140405 G3YCS1 FGSC A1144/LSHB Ac4/NCTC 3858a/NRRL 328/ USDA 3528.7) Bipolaris oryzae ATCC 44560 COCMIDRAFT_998 W7A2Q6 Cochliobolus heterostrophus (strain C4/ATCC 48331/ COCC4DRAFT_169587 N4X0M1 race T) (Southern corn leaf blight fungus) (Bipolaris maydis) Cochliobolus heterostrophus (strain C5/ATCC 48332/ COCHEDRAFT_1140715 M2U5K5 race O) (Southern corn leaf blight fungus) (Bipolaris maydis) Botryosphaeria parva (strain UCR-NP2) (Grapevine UCRNP2_24 R1GXQ4 canker fungus) (Neofusicoccum parvum) Ajellomyces capsulatus (strain H88) (Darling's disease HCEG_07709 F0URG7 fungus) (Histoplasma capsulatum) Bipolaris oryzae ATCC 44560 COCMIDRAFT_1291 W7A267 Dactylellina haptotyla (strain CBS 200.50) (Nematode- H072_402 S8ARJ7 trapping fungus) (Monacrosporium haptotylum) Cladophialophora carrionii CBS 160.54 G647_04218 V9DES7 Exophiala dermatitidis (strain ATCC 34100/CBS 525.76/ HMPREF1120_06284 H6C3Q7 NIH/UT8656) (Black yeast) (Wangiella dermatitidis) Yarrowia lipolytica (Candida lipolytica) ALK3 O74129 Yarrowia lipolytica (strain CLIB 122/E 150) (Yeast) YALI0_E23474g F2Z6D5 (Candida lipolytica) Blumeria graminis f. sp. hordei (strain DH14) (Barley BGHDH14_bgh01926 N1JHB2 powdery mildew) (Oidium monilioides f. sp. hordei) Neosartorya fischeri (strain ATCC 1020/DSM 3700/ NFIA_063580 A1D653 FGSC A1164/NRRL 181) (Aspergillus fischerianus) Dactylellina haptotyla (strain CBS 200.50) (Nematode- H072_3894 S8C337 trapping fungus) (Monacrosporium haptotylum) Aspergillus kawachii (strain NBRC 4308) (White koji AKAW_03269 G7XDZ6 mold) (Aspergillus awamori var. kawachi) Aspergillus niger (strain ATCC 1015/CBS 113.46/ ASPNIDRAFT_183349 G3Y6F0 FGSC A1144/LSHB Ac4/NCTC 3858a/NRRL 3287 USDA 3528.7) Aspergillus oryzae (strain 3.042) (Yellow koji mold) Ao3042_02280 I8IPH3 Aspergillus flavus (strain ATCC 200026/FGSC A1120/ AFLA_089870 B8NKB3 NRRL 3357/JCM 12722/SRRC 167) Arthroderma gypseum (strain ATCC MYA-4604/CBS MGYG_08468 E4V5T0 118893) (Microsporum gypseum) Arthroderma otae (strain ATCC MYA-4605/CBS MCYG_06305 C5FUA2 113480) (Microsporum canis) Bipolaris victoriae FI3 COCVIDRAFT_33397 W7EVM0 Bipolaris oryzae ATCC 44560 COCMIDRAFT_22726 W6ZD79 Byssochlamys spectabilis (strain No. 5/NBRC 109023) PVAR5_0072 V5HQF9 (Paecilomyces variotii) Bipolaris zeicola 26-R-13 COCCADRAFT_21391 W6YP58 Mycosphaerella fijiensis (strain CIRAD86) (Black leaf MYCFIDRAFT_153745 M3AEP8 streak disease fungus) (Pseudocercospora fijiensis) Aspergillus terreus (strain NIH 2624/FGSC A1156) ATEG_01150 Q0CYT4 Setosphaeria turcica (strain 28A) (Northern leaf blight SETTUDRAFT_96298 R0JZR2 fungus) (Exserohilum turcicum) Colletotrichum graminicola (strain M1.001/M2/FGSC GLRG_01676 E3Q5P1 10212) (Maize anthracnose fungus) (Glomerella graminicola) Aspergillus clavatus (strain ATCC 1007/CBS 513.65/ ACLA_055810 A1C9K9 DSM 816/NCTC 3887/NRRL 1) Ajellomyces capsulatus (strain G186AR/H82/ATCC HCBG_07070 C0NV90 MYA-2454/RMSCC 2432) (Darling's disease fungus) (Histoplasma capsulatum) Aspergillus oryzae (strain ATCC 42149/RIB 40) (Yellow AO090124000014 Q2U799 koji mold) Aspergillus oryzae (strain ATCC 42149/RIB 40) (Yellow AO090124000014 Q2U799 koji mold) Aspergillus niger (strain CBS 513.88/FGSC A1513) An11g04220 A2QW84 Penicillium marneffei (strain ATCC 18224/CBS 334.59/ PMAA_050330 B6QKF3 QM 7333) Tuber melanosporum (strain Mel28) (Perigord black GSTUM_00004620001 D5G7M1 truffle) Colletotrichum higginsianum (strain IMI 349063) CH063_01685 H1VAW5 (Crucifer anthracnose fungus) Beauveria bassiana (strain ARSEF 2860) (White BBA_08136 J5JHI3 muscardine disease fungus) (Tritirachium shiotae) Yarrowia lipolytica (strain CLIB 122/E 150) (Yeast) YALI0_C10054g Q6CCE5 (Candida lipolytica) Botryosphaeria parva (strain UCR-NP2) (Grapevine UCRNP2_3112 R1ERB7 canker fungus) (Neofusicoccum parvum) Setosphaeria turcica (strain 28A) (Northern leaf blight SETTUDRAFT_104406 R0IY48 fungus) (Exserohilum turcicum) Aspergillus clavatus (strain ATCC 1007/CBS 513.65/ ACLA_081330 A1CT08 DSM 816/NCTC 3887/NRRL 1) Mycosphaerella fijiensis (strain CIRAD86) (Black leaf MYCFIDRAFT_65755 M3A1E6 streak disease fungus) (Pseudocercospora fijiensis) Aspergillus oryzae (strain ATCC 42149/RIB 40) (Yellow AO090026000094 Q2UFS5 koji mold) Aspergillus oryzae (strain 3.042) (Yellow koji mold) Ao3042_06896 I7ZXQ6 Aspergillus oryzae (Yellow koji mold) CYP584G1 D4QC67 Aspergillus flavus (strain ATCC 200026/FGSC A1120/ AFLA_138460 B8NGX8 NRRL 3357/JCM 12722/SRRC 167) Candida tenuis (strain ATCC 10573/BCRC 21748/CBS CANTEDRAFT_120218 G3B201 615/JCM 9827/NBRC 10315/NRRL Y-1498/VKM Y-70) (Yeast) Aspergillus niger (strain ATCC 1015/CBS 113.46/ ASPNIDRAFT_51356 G3Y8H5 FGSC A1144/LSHB Ac4/NCTC 3858a/NRRL 3287 USDA 3528.7) Aspergillus niger (strain CBS 513.88/FGSC A1513) An03g02570 A2QGB4 Pseudogymnoascus destructans (strain ATCC MYA-4855/ GMDG_01087 L8FNT2 20631-21) (Bat white-nose syndrome fungus) (Geomyces destructans) Cladophialophora carrionii CBS 160.54 G647_06237 V9D5I6 Candida albicans (strain WO-1) (Yeast) CAWG_05505 C4YTL0 Coccidioides posadasii (strain RMSCC 757/Silveira) CPSG_05074 E9D644 (Valley fever fungus) Coccidioides posadasii (strain C735) (Valley fever fungus) CPC735_058630 C5PIZ0 Candida maltosa (strain Xu316) (Yeast) G210_3874 M3J212 Metarhizium acridum (strain CQMa 102) MAC_00168 E9DQZ9 Bipolaris zeicola 26-R-13 COCCADRAFT_112912 W6XW09 Pyronema omphalodes (strain CBS 100304) (Pyronema PCON_11087 U4LQK1 confluens) Bipolaris victoriae FI3 COCVIDRAFT_39745 W7E2W7 Botryotinia fuckeliana (strain T4) (Noble rot fungus) BofuT4_P153970.1 G2YW37 (Botrytis cinerea) Fusarium heterosporum fsdH S0ARX1 Cyphellophora europaea CBS 101466 HMPREF1541_04435 W2RWT0 Metarhizium acridum (strain CQMa 102) MAC_07120 E9EB72 Macrophomina phaseolina (strain MS6) (Charcoal rot MPH_05063 K2R5H7 fungus) Colletotrichum graminicola (strain M1.001/M2/FGSC GLRG_01883 E3Q8M4 10212) (Maize anthracnose fungus) (Glomerella graminicola) Bipolaris zeicola 26-R-13 COCCADRAFT_111835 W6Y8G6 Cochliobolus heterostrophus (strain C5/ATCC 48332/ COCHEDRAFT_1160314 M2SMR0 race O) (Southern corn leaf blight fungus) (Bipolaris maydis) Bipolaris zeicola 26-R-13 COCCADRAFT_101405 W6YJB0 Cochliobolus heterostrophus (strain C4/ATCC 48331/ COCC4DRAFT_62846 N4XCY6 race T) (Southern corn leaf blight fungus) (Bipolaris maydis) Colletotrichum gloeosporioides (strain Cg-14) CGLO_16193 T0L9W5 (Anthracnose fungus) (Glomerella cingulata) Botryotinia fuckeliana (strain BcDW1) (Noble rot fungus) BcDW1_1090 M7U9F3 (Botrytis cinerea) Botryotinia fuckeliana (strain T4) (Noble rot fungus) BofuT4P90000010001 G2YMJ6 (Botrytis cinerea) Sclerotinia sclerotiorum (strain ATCC 18683/1980/Ss-1) SS1G_10037 A7EXH2 (White mold) (Whetzelinia sclerotiorum) Penicillium digitatum (strain PHI26/CECT 20796) PDIG_44570 K9FT94 (Green mold) Penicillium digitatum (strain Pd1/CECT 20795) (Green PDIP_16560 K9GHJ2 mold) Metarhizium anisopliae (strain ARSEF 23/ATCC MYA-3075) MAA_06634 E9F2Y5 Starmerella bombicola B8QHP1 Penicillium marneffei (strain ATCC 18224/CBS 334.59/ PMAA_088180 B6QDT4 QM 7333) Metarhizium acridum (strain CQMa 102) MAC_09276 E9EHC8 Mycosphaerella pini (strain NZE10/CBS 128990) (Red DOTSEDRAFT_74860 N1PCY6 band needle blight fungus) (Dothistroma septosporum) Aspergillus kawachii (strain NBRC 4308) (White koji AKAW_10068 G7XYF8 mold) (Aspergillus awamori var. kawachi) Aspergillus niger (strain CBS 513.88/FGSC A1513) An13g03000 A2R1Z6 Aspergillus niger (strain ATCC 1015/CBS 113.46/ ASPNIDRAFT_44878 G3XQ89 FGSC A1144/LSHB Ac4/NCTC 3858a/NRRL 328/ USDA 3528.7) Beauveria bassiana (strain ARSEF 2860) (White BBA_05173 J4UM22 muscardine disease fungus) (Tritirachium shiotae) Beauveria bassiana (White muscardine disease fungus) E2EAF8 (Tritirachium shiotae) Aspergillus oryzae (strain 3.042) (Yellow koji mold) Ao3042_05622 I8IHV7 Aspergillus flavus (strain ATCC 200026/FGSC A1120/ AFLA_045270 B8NBF2 NRRL 3357/JCM 12722/SRRC 167) Aspergillus oryzae (strain ATCC 42149/RIB 40) (Yellow AO090011000712 Q2TZU9 koji mold) Aspergillus oryzae (Yellow koji mold) CYP52G3 D4QC12 Endocarpon pusillum (strain Z07020/HMAS-L-300199) EPUS_05482 U1GCZ9 (Lichen-forming fungus) Sclerotinia sclerotiorum (strain ATCC 18683/1980/Ss-1) SS1G_05980 A7EKY3 (White mold) (Whetzelinia sclerotiorum) Pyrenophora tritici-repentis (strain Pt-1C-BFP) (Wheat tan PTRG_06344 B2W8N6 spot fungus) (Drechslera tritici-repentis) Candida albicans (strain SC5314/ATCC MYA-2876) ALK6 CaO19.13927 Q5AGW4 (Yeast) CaO19.6574 Candida albicans (Yeast) ALK6 CaJ7.0170 G1U9Z0 CaO19.6574 Trichophyton verrucosum (strain HKI 0517) TRV_06353 D4DGP8 Coccidioides immitis (strain RS) (Valley fever fungus) CIMG_00331 J3KGS4 Ajellomyces dermatitidis ATCC 26199 BDFG_02901 T5C2N4 Ajellomyces dermatitidis (strain ATCC 18188/CBS BDDG_01558 F2T5V8 674.68) (Blastomyces dermatitidis) Ajellomyces dermatitidis (strain SLH14081) (Blastomyces BDBG_07037 C5JWU3 dermatitidis) Ajellomyces dermatitidis (strain ER-3/ATCC MYA-2586) BDCG_07223 C5GSH0 (Blastomyces dermatitidis) Coccidioides posadasii (strain C735) (Valley fever fungus) CPC735_073410 C5P014 Colletotrichum orbiculare (strain 104-T/ATCC 96160/ Cob_02045 N4V6W7 CBS 514.97/LARS 414/MAFF 240422) (Cucumber anthracnose fungus) (Colletotrichum lagenarium) Coccidioides immitis (strain RS) (Valley fever fungus) CIMG_11305 J3KDU2 Talaromyces stipitatus (strain ATCC 10500/CBS 375.48/ TSTA_056150 B8MRH9 QM 6759/NRRL 1006) (Penicillium stipitatum) Coccidioides posadasii (strain RMSCC 757/Silveira) CPSG_06231 E9D8S9 (Valley fever fungus) Uncinocarpus reesii (strain UAMH 1704) UREG_01634 C4JJ27 Starmerella bombicola B8QHP5 Pyrenophora tritici-repentis (strain Pt-1C-BFP) (Wheat tan PTRG_04241 B2W1A6 spot fungus) (Drechslera tritici-repentis) Marssonina brunnea f. sp. multigermtubi (strain MB_m1) MBM_07629 K1XPF9 (Marssonina leaf spot fungus) Metarhizium anisopliae (strain ARSEF 23/ATCC MYA-3075) MAA_00167 E9EKL9 Macrophomina phaseolina (strain MS6) (Charcoal rot MPH_02135 K2S0W5 fungus) Glarea lozoyensis (strain ATCC 20868/MF5171) GLAREA_00730 S3CV81 Arthroderma otae (strain ATCC MYA-4605/CBS MCYG_02969 C5FKC8 113480) (Microsporum canis) Trichophyton verrucosum (strain HKI 0517) TRV_03431 D4D8J5 Hypocrea atroviridis (strain ATCC 20476/IMI 206040) TRIATDRAFT_130690 G9P640 (Trichoderma atroviride) Glarea lozoyensis (strain ATCC 74030/MF5533) M7I_0305 H0ED06 Ajellomyces capsulatus (strain NAm1/WU24) (Darling's HCAG_08121 A6REQ6 disease fungus) (Histoplasma capsulatum) Pyronema omphalodes (strain CBS 100304) (Pyronema PCON_14046 U4LA29 confluens) Endocarpon pusillum (strain Z07020/HMAS-L-300199) EPUS_04540 U1GA45 (Lichen-forming fungus) Penicillium marneffei (strain ATCC 18224/CBS 334.59/ PMAA_048940 B6QS70 QM 7333) Emericella nidulans (strain FGSC A4/ATCC 38163/ AN6057.2 ANIA_06057 Q5B073 CBS 112.46/NRRL 194/M139) (Aspergillus nidulans) Botryotinia fuckeliana (strain T4) (Noble rot fungus) BofuT4_P148640.1 G2YX16 (Botrytis cinerea) Aspergillus terreus (strain NIH 2624/FGSC A1156) ATEG_02893 Q0CTU1 Aspergillus niger (strain ATCC 1015/CBS 113.46/ ASPNIDRAFT_55501 G3XTI8 FGSC A1144/LSHB Ac4/NCTC 3858a/NRRL 3287 USDA 3528.7) Aspergillus kawachii (strain NBRC 4308) (White koji AKAW_10154 G7XYN1 mold) (Aspergillus awamori var. kawachi) Beauveria bassiana (strain ARSEF 2860) (White BBA_02382 J4WEG4 muscardine disease fungus) (Tritirachium shiotae) Sclerotinia sclerotiorum (strain ATCC 18683/1980/Ss-1) SS1G_13470 A7F790 (White mold) (Whetzelinia sclerotiorum) Beauveria bassiana (strain ARSEF 2860) (White BBA_02428 J5K2V9 muscardine disease fungus) (Tritirachium shiotae) Beauveria bassiana (White muscardine disease fungus) E2EAF6 (Tritirachium shiotae) Cordyceps militaris (strain CM01) (Caterpillar fungus) CCM_04719 G3JD19 Penicillium chrysogenum (strain ATCC 28089/DSM Pc21g14130 B6HHN6 1075/Wisconsin 54-1255) (Penicillium notatum) PCH Pc21g14130 Mycosphaerella pini (strain NZE10/CBS 128990) (Red DOTSEDRAFT_70063 N1PRA2 band needle blight fungus) (Dothistroma septosporum) Mycosphaerella pini (strain NZE10/CBS 128990) (Red DOTSEDRAFT_70063 N1PRA2 band needle blight fungus) (Dothistroma septosporum) Aspergillus terreus (strain NIH 2624/FGSC A1156) ATEG_07540 Q0CFJ4 Arthroderma benhamiae (strain ATCC MYA-4681/CBS ARB_05989 D4AP22 112371) (Trichophyton mentagrophytes) Baudoinia compniacensis (strain UAMH 10762) (Angels' BAUCODRAFT_63612 M2NKX8 share fungus) Candida tropicalis (strain ATCC MYA-3404/T1) (Yeast) CTRG_04959 C5MFW6 Candida tropicalis (Yeast) CYP52C1 P30612 Metarhizium anisopliae (strain ARSEF 23/ATCC MYA-3075) MAA_07989 E9F6U0 Mycosphaerella graminicola (strain CBS 115943/ CYP-29MYCGRDRAFT_76681 F9XML6 IPO323) (Speckled leaf blotch fungus) (Septoria tritici) Cladophialophora carrionii CBS 160.54 G647_07950 V9D5N0 Glarea lozoyensis (strain ATCC 20868/MF5171) GLAREA_09137 S3DII6 Hypocrea virens (strain Gv29-8/FGSC 10586) TRIVIDRAFT_50878 G9N4V4 (Gliocladium virens) (Trichoderma virens) Marssonina brunnea f. sp. multigermtubi (strain MB_m1) MBM_02308 K1X1G5 (Marssonina leaf spot fungus) Talaromyces stipitatus (strain ATCC 10500/CBS 375.48/ TSTA_125560 B8MCM6 QM 6759/NRRL 1006) (Penicillium stipitatum) Arthroderma benhamiae (strain ATCC MYA-4681/CBS ARB_01737 D4AZW7 112371) (Trichophyton mentagrophytes) Colletotrichum higginsianum (strain IMI 349063) CH063_01286 H1V527 (Crucifer anthracnose fungus) Trichophyton tonsurans (strain CBS 112818) (Scalp TESG_03185 F2RW94 ringworm fungus) Marssonina brunnea f. sp. multigermtubi (strain MB_m1) MBM_09278 K1WJW9 (Marssonina leaf spot fungus) Aspergillus terreus (strain NIH 2624/FGSC A1156) ATEG_06678 Q0CI06 Claviceps purpurea (strain 20.1) (Ergot fungus) (Sphacelia CPUR_01906 M1VZT8 segetum) Trichophyton rubrum (strain ATCC MYA-4607/CBS TERG_01394 F2SCB2 118892) (Athlete's foot fungus) Setosphaeria turcica (strain 28A) (Northern leaf blight SETTUDRAFT_39981 R0ILM4 fungus) (Exserohilum turcicum) Paracoccidioides brasiliensis (strain Pb03) PABG_02000 C0S2T6 Arthroderma gypseum (strain ATCC MYA-4604/CBS MGYG_08184 E4V596 118893) (Microsporum gypseum) Trichophyton equinum (strain ATCC MYA-4606/CBS TEQG_06653 F2Q0K1 127.97) (Horse ringworm fungus) Talaromyces stipitatus (strain ATCC 10500/CBS 375.48/ TSTA_036210 B8M882 QM 6759/NRRL 1006) (Penicillium stipitatum) Leptosphaeria maculans (strain JN3/isolate v23.1.37 race LEMA_P030820.1 E4ZWF4 Av1-4-5-6-7-8) (Blackleg fungus) (Phoma lingam) Bipolaris victoriae FI3 COCVIDRAFT_43025 W7E4E8 Magnaporthe oryzae (strain Y34) (Rice blast fungus) OOU_Y34scaffold00740g4 L7HW63 (Pyricularia oryzae) Fusarium oxysporum f. sp. cubense (strain race 1) FOC1_g10015382 N4TN76 (Panama disease fungus) Fusarium oxysporum f. sp. lycopersici (strain 4287/CBS FOXG_00101 J9MB56 123668/FGSC 9935/NRRL 34936) (Fusarium vascular wilt of tomato) Endocarpon pusillum (strain Z07020/HMAS-L-300199) EPUS_06065 U1GKM5 (Lichen-forming fungus) Sphaerulina musiva (strain SO2202) (Poplar stem canker SEPMUDRAFT_55938 N1QLI3 fungus) (Septoria musiva) Mycosphaerella graminicola (strain CBS 115943/ CYP-31.1MYCGRDRAFT_47046 F9XH30 IPO323) (Speckled leaf blotch fungus) (Septoria tritici) Penicillium oxalicum (strain 114-2/CGMCC 5302) PDE_08994 S8BFY9 (Penicillium decumbens) Mycosphaerella graminicola (strain CBS 115943/ CYP-30MYCGRDRAFT_73230 F9XDL6 IPO323) (Speckled leaf blotch fungus) (Septoria tritici) Cladophialophora carrionii CBS 160.54 G647_01266 V9DPI5 Togninia minima (strain UCR-PA7) (Esca disease fungus) UCRPA7_6516 R8BF53 (Phaeoacremonium aleophilum) Fusarium oxysporum (strain Fo5176) (Fusarium vascular FOXB_00215 F9F1C9 wilt) Gaeumannomyces graminis var. tritici (strain R3-111a-1) GGTG_11345 J3PCX6 (Wheat and barley take-all root rot fungus) Cochliobolus sativus (strain ND90Pr/ATCC 201652) COCSADRAFT_143540 M2R997 (Common root rot and spot blotch fungus) (Bipolaris sorokiniana) Neosartorya fumigata (strain CEA10/CBS 144.89/ AFUB_002090 B0XRD5 FGSC A1163) (Aspergillus fumigatus) Neosartorya fumigata (strain ATCC MYA-4609/Af293/ AFUA_1G01690 Q4WKQ1 CBS 101355/FGSC A1100) (Aspergillus fumigatus) Neosartorya fischeri (strain ATCC 1020/DSM 3700/ NFIA_022940 A1D590 FGSC A1164/NRRL 181) (Aspergillus fischerianus) Hypocrea atroviridis (strain ATCC 20476/IMI 206040) TRIATDRAFT_239723 G9NQ55 (Trichoderma atroviride) Candida orthopsilosis (strain 90-125) (Yeast) CORT_0A06350 H8WY74 Cyphellophora europaea CBS 101466 HMPREF1541_09254 W2SBP3 Penicillium oxalicum (strain 114-2/CGMCC 5302) PDE_02656 S8B080 (Penicillium decumbens) Penicillium chrysogenum (strain ATCC 28089/DSM Pc20g13950 B6HH32 1075/Wisconsin 54-1255) (Penicillium notatum) PCH_Pc20g13950 Arthroderma gypseum (strain ATCC MYA-4604/CBS MGYG_07586 E4V3K6 118893) (Microsporum gypseum) Hypocrea virens (strain Gv29-8/FGSC 10586) TRIVIDRAFT_91340 G9MUE6 (Gliocladium virens) (Trichoderma virens) Botryotinia fuckeliana (strain BcDW1) (Noble rot fungus) BcDW1_3132 M7U3N6 (Botrytis cinerea) Botryosphaeria parva (strain UCR-NP2) (Grapevine UCRNP2_9778 R1E711 canker fungus) (Neofusicoccum parvum) Cochliobolus sativus (strain ND90Pr/ATCC 201652) COCSADRAFT_79461 M2SNB8 (Common root rot and spot blotch fungus) (Bipolaris sorokiniana) Aspergillus niger (strain CBS 513.88/FGSC A1513) An01g00510 A2Q7F5 Candida dubliniensis (strain CD36/ATCC MYA-646/ CD36_71370 B9WK39 CBS 7987/NCPF 3949/NRRL Y-17841) (Yeast) Cochliobolus heterostrophus (strain C4/ATCC 48331/ COCC4DRAFT_41710 N4XB06 race T) (Southern corn leaf blight fungus) (Bipolaris maydis) Cochliobolus heterostrophus (strain C5/ATCC 48332/ COCHEDRAFT_1208754 M2VA93 race O) (Southern corn leaf blight fungus) (Bipolaris maydis) Aspergillus clavatus (strain ATCC 1007/CBS 513.65/ ACLA_032820 A1CSC5 DSM 816/NCTC 3887/NRRL 1) Hypocrea jecorina (strain QM6a) (Trichoderma reesei) TRIREDRAFT_103147 G0R9K0 Trichophyton tonsurans (strain CBS 112818) (Scalp TESG_02758 F2RVB9 ringworm fungus) Glarea lozoyensis (strain ATCC 20868/MF5171) GLAREA_12102 S3D2G7 Trichophyton rubrum (strain ATCC MYA-4607/CBS TERG_03231 F2SJM4 118892) (Athlete's foot fungus) Leptosphaeria maculans (strain JN3/isolate v23.1.37 race LEMA_P073070.1 E5A7X3 Av1-4-5-6-7-8) (Blackleg fungus) (Phoma lingam) Cyphellophora europaea CBS 101466 HMPREF1541_04444 W2RWL1 Hypocrea jecorina (strain QM6a) (Trichoderma reesei) TRIREDRAFT_65036 G0RNX6 Beauveria bassiana (strain ARSEF 2860) (White BBA_09022 J5J6F5 muscardine disease fungus) (Tritirachium shiotae) Cordyceps militaris (strain CM01) (Caterpillar fungus) CCM_02084 G3JCK3 Trichophyton rubrum (strain ATCC MYA-4607/CBS TERG_05441 F2SSI7 118892) (Athlete's foot fungus) Botryotinia fuckeliana (strain BcDW1) (Noble rot fungus) BcDW1_9224 M7U6H3 (Botrytis cinerea) Magnaporthe oryzae (strain P131) (Rice blast fungus) OOW_P131scaffold01201g5 L7J0M9 (Pyricularia oryzae) Magnaporthe oryzae (strain Y34) (Rice blast fungus) OOU_Y34scaffold00145g13 L7IJZ9 (Pyricularia oryzae) Magnaporthe oryzae (strain 70-15/ATCC MYA-4617/ MGG_09920 G4MR75 FGSC 8958) (Rice blast fungus) (Pyricularia oryzae) Paracoccidioides lutzii (strain ATCC MYA-826/Pb01) PAAG_01378 C1GS83 (Paracoccidioides brasiliensis) Bipolaris zeicola 26-R-13 COCCADRAFT_9928 W6Y8S8 Verticillium dahliae (strain VdLs.17/ATCC MYA-4575/ VDAG_04483 G2X2F9 FGSC 10137) (Verticillium wilt) Trichophyton verrucosum (strain HKI 0517) TRV_02251 D4D581 Arthroderma benhamiae (strain ATCC MYA-4681/CBS ARB_01131 D4AY62 112371) (Trichophyton mentagrophytes) Chaetomium globosum (strain ATCC 6205/CBS 148.51/ CHGG_01610 Q2HDU4 DSM 1962/NBRC 6347/NRRL 1970) (Soil fungus) Magnaporthe poae (strain ATCC 64411/73-15) M4G6C3 (Kentucky bluegrass fungus) Hypocrea atroviridis (strain ATCC 20476/IMI 206040) TRIATDRAFT_45536 G9NQR1 (Trichoderma atroviride) Colletotrichum orbiculare (strain 104-T/ATCC 96160/ Cob_03064 N4W651 CBS 514.97/LARS 414/MAFF 240422) (Cucumber anthracnose fungus) (Colletotrichum lagenarium) Penicillium chrysogenum (strain ATCC 28089/DSM Pc20g11290 B6HG66 1075/Wisconsin 54-1255) (Penicillium notatum) PCH_Pc20g11290 Ophiocordyceps sinensis (strain Co18/CGMCC 3.14243) OCS_02874 T5AG58 (Yarsagumba caterpillar fungus) (Hirsutella sinensis) Pyrenophora teres f. teres (strain 0-1) (Barley net blotch PTT_07245 E3RH76 fungus) (Drechslera teres f. teres) Baudoinia compniacensis (strain UAMH 10762) (Angels' BAUCODRAFT_71913 M2MX22 share fungus) Podospora anserina (strain S/ATCC MYA-4624/DSM PODANS_0_160 B2AFV1 980/FGSC 10383) (Pleurage anserina) Aspergillus terreus (strain NIH 2624/FGSC A1156) ATEG_05807 Q0CKH7 Hypocrea jecorina (strain QM6a) (Trichoderma reesei) TRIREDRAFT_75713 G0RDE9 Claviceps purpurea (strain 20.1) (Ergot fungus) (Sphacelia CPUR_06997 M1WHP2 segetum) Aspergillus flavus (strain ATCC 200026/FGSC A1120/ AFLA_128090 B8NNJ8 NRRL 3357/JCM 12722/SRRC 167) Mycosphaerella fijiensis (strain CIRAD86) (Black leaf MYCFIDRAFT_49209 M3AV82 streak disease fungus) (Pseudocercospora fijiensis) Grosmannia clavigera (strain kw1407/UAMH 11150) CMQ_2882 F0XHG6 (Blue stain fungus) (Graphiocladiella clavigera) Lodderomyces elongisporus (strain ATCC 11503/CBS LELG_05768 A5H2Q3 2605/JCM 1781/NBRC 1676/NRRL YB-4239) (Yeast) (Saccharomyces elongisporus) Candida tropicalis (strain ATCC MYA-3404/T1) (Yeast) CTRG_03114 C5MAM2 Coniosporium apollinis (strain CBS 100218) (Rock- W97_03898 R7YRY7 inhabiting black yeast) Candida parapsilosis (strain CDC 317/ATCC MYA-4646) CPAR2_206990 G8BCR1 (Yeast) (Monilia parapsilosis) Aspergillus niger (strain CBS 513.88/FGSC A1513) An02g_10700 A5AAH7 Baudoinia compniacensis (strain UAMH 10762) (Angels' BAUCODRAFT_187941 M2MV99 share fungus) Candida tropicalis (Yeast) CYP52B1 P30611 Aspergillus kawachii (strain NBRC 4308) (White koji AKAW_04070 G7XG31 mold) (Aspergillus awamori var. kawachi) Colletotrichum gloeosporioides (strain Cg-14) CGLO_15455 T0JYY2 (Anthracnose fungus) (Glomerella cingulata) Colletotrichum gloeosporioides (strain Cg-14) CGLO_15455 T0JYY2 (Anthracnose fungus) (Glomerella cingulata) Endocarpon pusillum (strain Z07020/HMAS-L-300199) EPUS_09448 U1HSE1 (Lichen-forming fungus) Arthroderma gypseum (strain ATCC MYA-4604/CBS MGYG_00140 E5R368 118893) (Microsporum gypseum) Botryotinia fuckeliana (strain T4) (Noble rot fungus) BofuT4_P052870.1 G2XWR8 (Botrytis cinerea) Exophiala dermatitidis (strain ATCC 34100/CBS 525.76/ HMPREF1120_00302 H6BMQ6 NIH/UT8656) (Black yeast) (Wangiella dermatitidis) Aspergillus oryzae (Yellow koji mold) CYP52K1 D4QC15 Aspergillus oryzae (strain ATCC 42149/RIB 40) (Yellow AO090010000548 Q2TWI0 koji mold) Neurospora tetrasperma (strain FGSC 2508/ATCC NEUTE1DRAFT_150004 F8N2K8 MYA-4615/P0657) Sordaria macrospora (strain ATCC MYA-333/DSM 997/ SMAC_04611 F7W1Z0 K(L3346)/K-hell) Neurospora crassa (strain ATCC 24698/74-OR23-1A/ NCU09115 Q7S0G0 CBS 708.71/DSM 1257/FGSC 987) Eutypa lata (strain UCR-EL1) (Grapevine dieback disease UCREL1_11542 M7T4H1 fungus) (Eutypa armeniacae) Neurospora tetrasperma (strain FGSC 2509/P0656) NEUTE2DRAFT_153986 G4U5S9 Setosphaeria turcica (strain 28A) (Northern leaf blight SETTUDRAFT_164879 R0JQZ4 fungus) (Exserohilum turcicum) Pyrenophora tritici-repentis (strain Pt-1C-BFP) (Wheat tan PTRG_11480 B2WN31 spot fungus) (Drechslera tritici-repentis) Paracoccidioides lutzii (strain ATCC MYA-826/Pb01) PAAG_01137 C1GRJ2 (Paracoccidioides brasiliensis) Neosartorya fischeri (strain ATCC 1020/DSM 3700/ NFIA_054110 A1DMP4 FGSC A1164/NRRL 181) (Aspergillus fischerianus) Sphaerulina musiva (strain SO2202) (Poplar stem canker SEPMUDRAFT_149283 M3D461 fungus) (Septoria musiva) Emericella nidulans (strain FGSC A4/ATCC 38163/ AN3917.2 ANIA_03917 Q5B6B3 CBS 112.46/NRRL 194/M139) (Aspergillus nidulans) Candida orthopsilosis (strain 90-125) (Yeast) CORT_0H01020 H8XAX0 Aspergillus oryzae (strain 3.042) (Yellow koji mold) Ao3042_10143 I8I9N9 Aspergillus flavus (strain ATCC 200026/FGSC A1120/ AFLA_074560 B8MWJ8 NRRL 3357/JCM 12722/SRRC 167) Candida parapsilosis (strain CDC 317/ATCC MYA-4646) CPAR2_301000 G8B912 (Yeast) (Monilia parapsilosis) Aspergillus oryzae (strain ATCC 42149/RIB 40) (Yellow AO090005000220 Q2UT03 koji mold) Aspergillus oryzae (Yellow koji mold) CYP52G4 D4QC13 Neosartorya fumigata (strain ATCC MYA-4609/Af293/ AFUA_6G03090 Q4WD09 CBS 101355/FGSC A1100) (Aspergillus fumigatus) Botryotinia fuckeliana (strain T4) (Noble rot fungus) BofuT4_P109530.1 G2Y7G7 (Botrytis cinerea) Sclerotinia sclerotiorum (strain ATCC 18683/1980/Ss-1) SS1G_11430 A7F1G0 (White mold) (Whetzelinia sclerotiorum) Pyronema omphalodes (strain CBS 100304) (Pyronema PCON_02181 U4LWN1 confluens) Thielavia heterothallica (strain ATCC 42464/BCRC MYCTH_2294752 G2Q2L5 31852/DSM 1799) (Myceliophthora thermophila) Pestalotiopsis fici W106-1 PFICI_00042 W3XJN2 Eutypa lata (strain UCR-EL1) (Grapevine dieback disease UCREL1_5311 M7TLT9 fungus) (Eutypa armeniacae) Colletotrichum orbiculare (strain 104-T/ATCC 96160/ Cob_03446 N4W590 CBS 514.97/LARS 414/MAFF 240422) (Cucumber anthracnose fungus) (Colletotrichum lagenarium) Colletotrichum graminicola (strain M1.001/M2/FGSC GLRG_09839 E3QV05 10212) (Maize anthracnose fungus) (Glomerella graminicola) Trichophyton verrucosum (strain HKI 0517) TRV_06704 D4DHP8 Sphaerulina musiva (strain SO2202) (Poplar stem canker SEPMUDRAFT_39329 M3B7G5 fungus) (Septoria musiva) Nectria haematococca (strain 77-13-4/ATCC MYA-4622/ NECHADRAFT_31103 C7YK50 FGSC 9596/MPVI) (Fusarium solani subsp. pisi) Coniosporium apollinis (strain CBS 100218) (Rock- W97_03080 R7YPM4 inhabiting black yeast) Gaeumannomyces graminis var. tritici (strain R3-111a-1) GGTG_12245 J3PFH0 (Wheat and barley take-all root rot fungus) Fusarium pseudograminearum (strain CS3096) (Wheat FPSE_11595 K3V5M1 and barley crown-rot fungus) Magnaporthe oryzae (strain P131) (Rice blast fungus) OOW_P131scaffold00556g2 L7J9P9 (Pyricularia oryzae) Magnaporthe oryzae (strain Y34) (Rice blast fungus) OOU_Y34scaffold00501g3 L7I9Z3 (Pyricularia oryzae) Magnaporthe oryzae (strain 70-15/ATCC MYA-4617/ MGG_08956 G4MW35 FGSC 8958) (Rice blast fungus) (Pyricularia oryzae) Thielavia terrestris (strain ATCC 38088/NRRL 8126) THITE_2057357 G2RF28 (Acremonium alabamense) Gibberella fujikuroi (strain CBS 195.34/IMI 58289/ FFUJ_01480 S0DIN1 NRRL A-6831) (Bakanae and foot rot disease fungus) (Fusarium fujikuroi) Pyronema omphalodes (strain CBS 100304) (Pyronema PCON_09796 U4L3P6 confluens) Gibberella moniliformis (strain M3125/FGSC 7600) FVEG_01415 W7LF29 (Maize ear and stalk rot fungus) (Fusarium verticillioides) Magnaporthe oryzae (strain P131) (Rice blast fungus) OOW_P131scaffold01216g6 L7IZ69 (Pyricularia oryzae) Magnaporthe oryzae (strain 70-15/ATCC MYA-4617/ MGG_08494 G4NAN9 FGSC 8958) (Rice blast fungus) (Pyricularia oryzae) Fusarium oxysporum f. sp. cubense (strain race 4) FOC4_g10003027 N1RRF1 (Panama disease fungus) Chaetomium thermophilum (strain DSM 1495/CBS CTHT_0057700 G0SCL9 144.50/IMI 039719) Botryotinia fuckeliana (strain BcDW1) (Noble rot fungus) BcDW1_5818 M7UFT7 (Botrytis cinerea) Verticillium alfalfae (strain VaMs.102/ATCC MYA-4576/ VDBG_04942 C9SIR0 FGSC 10136) (Verticillium wilt of alfalfa) (Verticillium albo-atrum) Arthroderma gypseum (strain ATCC MYA-4604/CBS MGYG_09210 E4V712 118893) (Microsporum gypseum) Uncinocarpus reesii (strain UAMH 1704) UREG_00942 C4JF41 Bipolaris oryzae ATCC 44560 COCMIDRAFT_34506 W6ZKE3 Paracoccidioides brasiliensis (strain Pb03) PABG_01712 C0S297 Paracoccidioides brasiliensis (strain Pb18) PADG_03693 C1G8V7 Neosartorya fumigata (strain ATCC MYA-4609/Af293/ AFUA_6G08460 Q4WMW7 CBS 101355/FGSC A1100) (Aspergillus fumigatus) Neosartorya fumigata (strain CEA10/CBS 144.89/ AFUB_074420 B0Y7N4 FGSC A1163) (Aspergillus fumigatus) Aspergillus niger (strain ATCC 1015/CBS 113.46/ ASPNIDRAFT_189129 G3XM79 FGSC A1144/LSHB Ac4/NCTC 3858a/NRRL 328/ USDA 3528.7) Coniosporium apollinis (strain CBS 100218) (Rock- W97_05529 R7YX00 inhabiting black yeast) Aspergillus niger (strain CBS 513.88/FGSC A1513) An11g07010 A2QWZ5 Aspergillus niger (strain ATCC 1015/CBS 113.46/ ASPNIDRAFT_56022 G3YAT8 FGSC A1144/LSHB Ac4/NCTC 3858a/NRRL 328/ USDA 3528.7) Neosartorya fischeri (strain ATCC 1020/DSM 3700/ NFIA_049440 A1DLD3 FGSC A1164/NRRL 181) (Aspergillus fischerianus) Aspergillus oryzae (strain ATCC 42149/RIB 40) (Yellow AO090010000075 Q2TXN5 koji mold) Aspergillus oryzae (strain 3.042) (Yellow koji mold) Ao3042_00404 I8ABC4 Aspergillus oryzae (Yellow koji mold) CYP584E5 D4QC66 Magnaporthe poae (strain ATCC 64411/73-15) M4GA78 (Kentucky bluegrass fungus) Cladophialophora carrionii CBS 160.54 G647_04914 V9DAX0 Podospora anserina (strain S/ATCC MYA-4624/DSM PODANS_1_9520 B2AY12 980/FGSC 10383) (Pleurage anserina) Gibberella zeae (strain PH-1/ATCC MYA-4620/FGSC FG01284.1 FGSG_01284 I1RCH0 9075/NRRL 31084) (Wheat head blight fungus) (Fusarium graminearum) Colletotrichum orbiculare (strain 104-T/ATCC 96160/ Cob_05738 N4VDA5 CBS 514.97/LARS 414/MAFF 240422) (Cucumber anthracnose fungus) (Colletotrichum lagenarium) Neosartorya fischeri (strain ATCC 1020/DSM 3700/ NFIA_113870 A1D8Z5 FGSC A1164/NRRL 181) (Aspergillus fischerianus) Trichophyton verrucosum (strain HKI 0517) TRV_00955 D4D1K4 Botryotinia fuckeliana (strain T4) (Noble rot fungus) BofuT4_P140470.1 G2YYT7 (Botrytis cinerea) Trichophyton rubrum (strain ATCC MYA-4607/CBS TERG_02747 F2SKM8 118892) (Athlete's foot fungus) Botryotinia fuckeliana (strain T4) (Noble rot fungus) BofuT4_P075800.1 G2XNP1 (Botrytis cinerea) Setosphaeria turcica (strain 28A) (Northern leaf blight SETTUDRAFT_159435 R0IYM1 fungus) (Exserohilum turcicum) Bipolaris victoriae FI3 COCVIDRAFT_96117 W7ECV3 Bipolaris zeicola 26-R-13 COCCADRAFT_86052 W6YCN0 Podospora anserina (strain S/ATCC MYA-4624/DSM PODANS_3_ 1920 B2AZX1 980/FGSC 10383) (Pleurage anserina) Sporothrix schenckii (strain ATCC 58251/de Perez HMPREF1624_01101 U7Q4H5 2211183) (Rose-picker's disease fungus) Exophiala dermatitidis (strain ATCC 34100/CBS 525.76/ HMPREF1120_04188 H6BWM7 NIH/UT8656) (Black yeast) (Wangiella dermatitidis) Colletotrichum gloeosporioides (strain Cg-14) CGLO_16096 T0JPF3 (Anthracnose fungus) (Glomerella cingulata) Arthroderma benhamiae (strain ATCC MYA-4681/CBS ARB_05099 D4ALA2 112371) (Trichophyton mentagrophytes) Macrophomina phaseolina (strain MS6) (Charcoal rot MPH_10488 K2QR42 fungus) Trichophyton tonsurans (strain CBS 112818) (Scalp TESG_05856 F2S4I4 ringworm fungus) Trichophyton equinum (strain ATCC MYA-4606/CBS TEQG_04559 F2PUI2 127.97) (Horse ringworm fungus) Arthroderma benhamiae (strain ATCC MYA-4681/CBS ARB_07892 D4AUH5 112371) (Trichophyton mentagrophytes) Arthroderma otae (strain ATCC MYA-4605/CBS MCYG_08648 C5G126 113480) (Microsporum canis) Aspergillus flavus (strain ATCC 200026/FGSC A1120/ AFLA_116530 B8NVG6 NRRL 3357/JCM 12722/SRRC 167) Mycosphaerella graminicola (strain CBS 115943/ CYP-28MYCGRDRAFT_111399 F9XPH9 IPO323) (Speckled leaf blotch fungus) (Septoria tritici) Penicillium chrysogenum (strain ATCC 28089/DSM Pc18g04990 B6HBW9 1075/Wisconsin 54-1255) (Penicillium notatum) PCH_Pc18g04990 Alternaria solani alt2 Q5KTN2 Colletotrichum higginsianum (strain IMI 349063) CH063_05380 H1UYS7 (Crucifer anthracnose fungus) Thielavia heterothallica (strain ATCC 42464/BCRC MYCTH_2060315 G2QDC4 31852/DSM 1799) (Myceliophthora thermophila) Togninia minima (strain UCR-PA7) (Esca disease fungus) UCRPA7_1480 R8BUP2 (Phaeoacremonium aleophilum) Ophiostoma piceae (strain UAMH 11346) (Sap stain F503_00556 S3C2T4 fungus) Cladophialophora carrionii CBS 160.54 G647_02236 V9DGM2 Botryotinia fuckeliana (strain BcDW1) (Noble rot fungus) BcDW1_141 M7UBZ7 (Botrytis cinerea) Mycobacterium sp. HXN-1500 cyp153 Q65A64 Gordonia amicalis NBRC 100051 = JCM 11271 GOAMI_64_00090 L7L6P4 Mycobacterium austroafricanum B6UKY3 Mycobacterium sp. ENV421 ahpG I7CD96 uncultured bacterium cyp153 W0UDE1 uncultured bacterium P450 Q33DR8 uncultured bacterium P450 Q33DR9 uncultured bacterium cyp153 W0UDG2 uncultured bacterium cyp153 W0UDM1 uncultured bacterium cyp153 W0UCX8 uncultured bacterium cyp153 W0UAP1 uncultured bacterium cyp153 W0UCW9 Polaromonas sp. (strain JS666/ATCC BAA-500) Bpro_5301 Q11ZY2 uncultured bacterium cyp153 W0UDK1 uncultured bacterium cyp153 W0UD29 uncultured bacterium cyp153 W0UD32 uncultured bacterium cyp153 W0UD27 uncultured bacterium cyp153 W0UAW2 uncultured bacterium cyp153 W0UAW6 Parvibaculum sp. S13-6 CYP153A C7A8P8 uncultured bacterium cyp153 W0UDM5 uncultured bacterium cyp153 W0UD31 uncultured bacterium cyp153 W0UDB6 Parvibaculum sp. S13-5 CYP153A C7A8P2 uncultured bacterium P450 Q33DS1 uncultured bacterium cyp153 W0UDK5 uncultured bacterium cyp153 W0UDU1 Tistrella mobilis CYP153A C7A8Q6 uncultured bacterium cyp153 W0UDS7 Parvibaculum sp. S13-6 CYP153A C7A8P9 uncultured bacterium cyp153 W0UB47 Parvibaculum sp. S13-6 CYP153A C7A8P7 gamma proteobacterium S10-1 CYP153A C7A8N2 uncultured bacterium cyp153 W0UDS4 uncultured bacterium cyp153 W0UAY8 uncultured bacterium cyp153 W0UDB2 uncultured bacterium cyp153 W0UB02 uncultured bacterium cyp153 W0UDV5 uncultured bacterium cyp153 W0UDM7 uncultured bacterium cyp153 W0UD83 uncultured bacterium cyp153 W0UD50 Parvibaculum sp. S13-5 CYP153A C7A8P4 Parvibaculum sp. S18-4 CYP153A C7A8S8 Parvibaculum sp. S18-4 CYP153A C7A8S9 uncultured bacterium cyp153 W0UB69 Parvibaculum sp. S13-5 CYP153A C7A8P5 uncultured bacterium cyp153 W0UDU6 uncultured bacterium cyp153 W0UDD0 uncultured bacterium cyp153 W0UDA8 uncultured bacterium cyp153 W0UDC3 uncultured bacterium cyp153 W0UDF5 uncultured bacterium cyp153 W0UDD2 uncultured bacterium cyp153 W0UD99 uncultured bacterium cyp153 W0UB78 uncultured bacterium cyp153 W0UDU2 uncultured bacterium cyp153 W0UD95 uncultured bacterium cyp153 W0UDT1 uncultured bacterium cyp153 W0UD70 uncultured bacterium cyp153 W0UAV3 uncultured bacterium cyp153 W0UDJ0 Parvibaculum sp. S18-4 CYP153A C7A8S7 uncultured bacterium cyp153 W0UD49 uncultured bacterium cyp153 W0UB74 uncultured bacterium cyp153 W0UDG4 uncultured bacterium cyp153 W0UDJ4 uncultured bacterium cyp153 W0UDL1 uncultured bacterium cyp153 W0UD80 uncultured bacterium cyp153 W0UDP8 uncultured bacterium cyp153 W0UDS6 uncultured bacterium cyp153 W0UDC9 uncultured bacterium cyp153 W0UDE6 uncultured bacterium cyp153 W0UDU9 uncultured bacterium cyp153 W0UDC0 uncultured bacterium cyp153 W0UDW1 uncultured bacterium cyp153 W0UDT4 uncultured bacterium cyp153 W0UDB5 uncultured bacterium cyp153 W0UB64 uncultured bacterium cyp153 W0UDA3 uncultured bacterium cyp153 W0UDR7 uncultured bacterium cyp153 W0UB52 uncultured bacterium cyp153 W0UDA5 uncultured bacterium cyp153 W0UDT6 Caulobacter sp. (strain K31) Caul_0020 B0T154 uncultured bacterium cyp153 W0UCV6 uncultured bacterium cyp153 W0UCU1 uncultured bacterium cyp153 W0UDK0 uncultured bacterium cyp153 W0UDI6 uncultured bacterium cyp153 W0UAU9 uncultured bacterium cyp153 W0UAZ2 uncultured bacterium cyp153 W0UD75 uncultured bacterium cyp153 W0UD14 uncultured bacterium cyp153 W0UB97 uncultured bacterium cyp153 W0UD23 uncultured bacterium cyp153 W0UD18 uncultured bacterium cyp153 W0UDQ2 uncultured bacterium cyp153 W0UDH4 uncultured bacterium cyp153 W0UAT6 uncultured bacterium cyp153 W0UD79 uncultured bacterium cyp153 W0UAN4 uncultured bacterium cyp153 W0UDW9 uncultured bacterium cyp153 W0UCZ3 uncultured bacterium cyp153 W0UCZ3 Erythrobacter sp. S11-13 CYP153A C7A8R4 uncultured bacterium cyp153 W0UDK7 Parvibaculum sp. S13-5 CYP153A C7A8P3 uncultured bacterium cyp153 W0UDS2 uncultured bacterium cyp153 W0UD84 uncultured bacterium cyp153 W0UD90 uncultured bacterium cyp153 W0UB38 uncultured bacterium cyp153 W0UCW4 uncultured bacterium cyp153 W0UB22 uncultured bacterium cyp153 W0UDQ8 uncultured Rhizobiales bacterium HF4000_48A13 E0XZ55 uncultured Rhizobiales bacterium HF4000_48A13 E0XZ44 uncultured bacterium P450 Q33DS2 uncultured bacterium P450 Q33DS0 uncultured bacterium cyp153 W0UDB4 Erythrobacter flavus C5MKK1 uncultured bacterium cyp153 W0UD08 uncultured bacterium cyp153 W0UCW2 Sphingobium sp. S13-2 CYP153A C7A8P1 Sphingopyxis sp. S16-14 CYP153A C7A8R8 uncultured bacterium cyp153 W0UD46 Parvibaculum sp. S13-6 CYP153A C7A8P6 uncultured bacterium cyp153 W0UDQ1 uncultured bacterium cyp153 W0UB27 uncultured bacterium cyp153 W0UD73 uncultured bacterium cyp153 W0UDE2 uncultured bacterium cyp153 W0UD17 Erythrobacter sp. S17-1 CYP153A C7A8R9 uncultured bacterium cyp153 W0UD15 uncultured bacterium cyp153 W0UAU6 Erythrobacter flavus CYP153A C7A8N4 uncultured bacterium cyp153 W0UDD6 uncultured bacterium cyp153 W0UDP1 uncultured bacterium cyp153 W0UDF8 uncultured bacterium cyp153 W0UDN8 uncultured bacterium cyp153 W0UDD3 uncultured bacterium cyp153 W0UDN1 uncultured bacterium cyp153 W0UDK3 uncultured bacterium cyp153 W0UD11 uncultured bacterium cyp153 W0UB85 uncultured bacterium cyp153 W0UDI2 Bradyrhizobium sp. CCGE-LA001 BCCGELA001_36078 W1JJD5 uncultured bacterium cyp153 W0UDP5 uncultured bacterium cyp153 W0UB19 uncultured bacterium cyp153 W0UAL6 uncultured bacterium cyp153 W0UDN3 uncultured bacterium cyp153 W0UD72 uncultured bacterium cyp153 W0UCX1 uncultured bacterium cyp153 W0UDF6 uncultured bacterium cyp153 W0UD00 uncultured bacterium cyp153 W0UD65 Caulobacter sp. AP07 PMI01_00728 J2H335 Parvibaculum lavamentivorans (strain DS-1/DSM 13023/ Plav_1765 A7HU01 NCIMB 13966) uncultured bacterium P450 Q33DS3 uncultured bacterium cyp153 W0UDH8 Erythrobacter flavus CYP153A C7A8R2 Erythrobacter sp. S2-1 CYP153A C7A8K9 Erythrobacter citreus CYP153A C7A8R1 Erythrobacter citreus CYP153A C7A8R3 Erythrobacter flavus CYP153A C7A8N5 uncultured bacterium cyp153 W0UD37 Erythrobacter sp. S14-1 CYP153A C7A8Q4 uncultured bacterium cyp153 W0UDF2 uncultured bacterium cyp153 W0UDR6 uncultured bacterium cyp153 W0UAN1 uncultured bacterium cyp153 W0UCX5 uncultured bacterium cyp153 W0UD38 uncultured bacterium cyp153 W0UDM9 uncultured bacterium cyp153 W0UCW7 uncultured bacterium cyp153 W0UB12 uncultured bacterium cyp153 W0UD04 uncultured bacterium cyp153 W0UDQ6 Sphingopyxis macrogoltabida (Sphingomonas ahpG1 Q5F4D9 macrogoltabidus) Afipia broomeae ATCC 49717 HMPREF9695_03199 K8P5Q2 uncultured bacterium cyp153 W0UD96 Parvibaculum sp. S18-4 CYP153A C7A8S5 uncultured bacterium cyp153 W0UAN7 uncultured bacterium cyp153 W0UCS9 uncultured bacterium cyp153 W0UDX6 uncultured bacterium cyp153 W0UDB7 uncultured bacterium cyp153 W0UD56 uncultured bacterium cyp153 W0UD44 Parvibaculum lavamentivorans (strain DS-1/DSM 13023/ Plav_2128 A7HV09 NCIMB 13966) Caulobacter crescentus (strain NA1000/CB15N) CCNA_00061 B8GXF2 Caulobacter crescentus (strain ATCC 19089/CB15) CC_0063 Q9AC06 Parvibaculum lavamentivorans (strain DS-1/DSM 13023/ Plav_0025 A7HP15 NCIMB 13966) Caulobacter segnis (strain ATCC 21756/DSM 7131/ Cseg_0011 D5VDJ3 JCM 7823/NBRC 15250/LMG 17158/TK0059) (Mycoplana segnis) Novosphingobium sp. PP1Y PP1Y_AT31178 F6IH26 uncultured bacterium cyp153 W0UDC7 uncultured bacterium cyp153 W0UDA2 uncultured bacterium cyp153 W0UDP7 Parvibaculum sp. S18-4 CYP153A C7A8S6 uncultured bacterium cyp153 W0UAK6 uncultured bacterium cyp153 W0UD52 uncultured bacterium cyp153 W0UCU6 uncultured bacterium cyp153 W0UCR4 uncultured bacterium cyp153 W0UCS6 uncultured bacterium cyp153 W0UDV6 uncultured bacterium cyp153 W0UDY0 uncultured bacterium cyp153 W0UDF0 uncultured bacterium cyp153 W0UDF0 uncultured bacterium cyp153 W0UAV7 uncultured bacterium cyp153 W0UDL7 Bradyrhizobium sp. STM 3843 BRAS3843_1530026 H0THQ7 Bradyrhizobium sp. (strain ORS278) BRADO1446 A4YN62 Bradyrhizobium sp. (strain BTAi1/ATCC BAA-1182) BBta_6659 A5EQW5 Caulobacter crescentus OR37 OR37_01714 R0EKG8 Afipia broomeae ATCC 49717 HMPREF9695_03200 K8P2K6 Afipia clevelandensis ATCC 49720 HMPREF9696_02236 K8P5K9 Bradyrhizobiaceae bacterium SG-6C CSIRO_4275 F7QRQ2 Novosphingobium pentaromativorans US6-1 ahpG3 NSU_pLA1167 G6EL94 marine gamma proteobacterium HTCC2143 GP2143_12206 A0YHG8 Sphingopyxis macrogoltabida (Sphingomonas ahpG2 Q5F4D6 macrogoltabidus) uncultured bacterium cyp153 W0UD98 uncultured bacterium cyp153 W0UAZ7 uncultured bacterium cyp153 W0UCU0 uncultured bacterium cyp153 W0UCW6 Bradyrhizobium sp. ORS 375 BRAO375_960079 H0SSR8 Bradyrhizobium sp. ORS 285 BRAO285_1310010 H0RSU1 Bradyrhizobium sp. STM 3809 BRAS3809_1790009 H0SVY3 Rhodopseudomonas palustris (strain BisA53) RPE_4309 Q07IK1 Bradyrhizobium sp. YR681 PMI42_06128 J3CQJ7 Bradyrhizobium sp. STM 3843 BRAS3843_1530027 H0THQ8 Rhodopseudomonas palustris (strain BisB18) RPC_4264 Q20YJ8 Caulobacter sp. (strain K31) Caul_5296 B0T9L7 Sphingopyxis macrogoltabida (Sphingomonas ahpG3 Q5F4D3 macrogoltabidus) Bradyrhizobium oligotrophicum S58 S58_15720 M4ZMZ3 Bradyrhizobium diazoefficiens (strain JCM 10833/IAM blr7242 Q89E45 13628/NBRC 14792/USDA 110) uncultured bacterium cyp153 W0UAK0 uncultured bacterium cyp153 W0UD34 Bradyrhizobium oligotrophicum S58 S58_15730 M4Z3Y5 Erythrobacter litoralis (strain HTCC2594) ELI_14945 Q2N5G0 Erythrobacter sp. SD-21 ED21_32074 A5PDG4 Bradyrhizobium sp. DFCI-1 C207_05440 U1GV14 Bradyrhizobium sp. DFCI-1 C207_05439 U1HA94 Bradyrhizobium diazoefficiens (strain JCM 10833/IAM blr7243 Q89E44 13628/NBRC 14792/USDA 110) Rhodopseudomonas palustris (strain TIE-1) Rpa1_1803 B3Q8D0 Bradyrhizobium sp. CCGE-LA001 BCCGELA001_36088 W1JKM5 Parvibaculum lavamentivorans (strain DS-1/DSM 13023/ Plav_1782 A7HU17 NCIMB 13966) Rhodopseudomonas palustris (strain ATCC BAA-98/ RPA1613 Q6N9D6 CGA009) Bradyrhizobium sp. S23321 S23_58660 I0GE69 Bradyrhizobium sp. ORS 285 BRAO285_1310011 H0RSU2 Bradyrhizobium sp. ORS 375 BRAO375_960081 H0SSR9 Bradyrhizobium sp. (strain BTAi1/ATCC BAA-1182) BBta_6660 A5EQW6 Bradyrhizobium japonicum USDA 6 BJ6T_79720 G7DEP2 uncultured bacterium cyp153 W0UDA7 uncultured bacterium cyp153 W0UDB9 Afipia sp. P52-10 X566_03415 W3RJ54 Afipia sp. P52-10 X566_20970 W3RG92 marine gamma proteobacterium HTCC2143 GP2143_06774 A0YGV8 Afipia sp. P52-10 X566_16815 W3RJ04 Bradyrhizobium japonicum USDA 6 BJ6T_21500 G7D7D2 Bradyrhizobium sp. WSM471 Bra471DRAFT_06475 H5YKH9 Bradyrhizobium sp. S23321 S23_58670 I0GE70 Rhodopseudomonas palustris (strain DX-1) Rpdx1_3910 E6VIP2 Bradyrhizobium sp. STM 3809 BRAS3809_1790008 H0SVY2 Bradyrhizobium sp. (strain ORS278) BRADO1445 A4YN61 Rhodopseudomonas palustris (strain HaA2) RPB_3934 Q2IT33 Rhodopseudomonas palustris (strain BisBS) RPD_3694 Q132S4 Phenylobacterium zucineum (strain HLK1) p450 PHZ_c0813 B4RGA3 Bradyrhizobium sp. WSM1253 Bra1253DRAFT_03743 I2QGW7 Bradyrhizobium sp. WSM471 Bra471DRAFT_06476 H5YKI0 Bradyrhizobium sp. WSM1253 Bra1253DRAFT_03744 I2QGW8 Bradyrhizobium japonicum USDA 6 BJ6T_21490 G7D7D1 Bradyrhizobium sp. YR681 PMI42_06129 J2WD32 Afipia sp. P52-10 X566_20975 W3RG20 gamma proteobacterium NOR5-3 NOR53_2355 B8KH72 Bradyrhizobium sp. CCGE-LA001 BCCGELA001_12206 W1JZ89 marine gamma proteobacterium HTCC2148 GPB2148_2599 B7RZN8 gamma proteobacterium BDW918 DOK_00120 I2JQ45 Congregibacter litoralis KT71 KT71_14444 A4A7Y2 Bradyrhizobium diazoefficiens (strain JCM 10833/IAM blr1853 H7C6Q5 13628/NBRC 14792/USDA 110) Bradyrhizobium japonicum id311 Q9AND6 uncultured bacterium cyp153 W0UCV0 uncultured bacterium cyp153 W0UAD7 Pseudomonas sp. 19-rlim G3LGZ6 Bradyrhizobium sp. WSM1253 Bra1253DRAFT_06024 I2QN59 Bradyrhizobium sp. WSM471 Bra471DRAFT_01541 H5Y7S1 uncultured gamma proteobacterium EB000_65A11 E0XZZ2 marine gamma proteobacterium HTCC2148 GPB2148_1452 B7RXX8 marine gamma proteobacterium HTCC2143 GP2143_15156 A0Y901 Afipia sp. P52-10 X566_17435 W3RGW1 gamma proteobacterium NOR5-3 NOR53_537 B8KPR5 Glaciecola psychrophila 170 C427_3047 GPSY_3092 K7ADG3 Marinobacter lipolyticus SM19 MARLIPOL_15764 R8AWZ8 gamma proteobacterium IMCC3088 IMCC3088_2432 F3L451 uncultured bacterium P450 Q33DT3 uncultured bacterium P450 Q33DS9 uncultured bacterium P450 Q33DS8 uncultured bacterium cyp153 W0UD71 Congregibacter litoralis KT71 KT71_02837 A4A779 marine gamma proteobacterium HTCC2080 MGP2080_14441 A0Z7J1 Marinobacter santoriniensis NKSG1 MSNKSG1_10343 M7CRK4 Alcanivorax hongdengensis G1C7P2 Alcanivorax sp. DG881 ADG881_2620 B4WXL2 uncultured bacterium P450 Q33DS6 uncultured bacterium cyp153 W0UCP6 uncultured bacterium cyp153 W0UCQ6 Ochrobactrum anthropi CYP153A C7A8M0 uncultured bacterium cyp153 W0UCN8 uncultured bacterium cyp153 W0UCT1 uncultured bacterium cyp153 W0UCT1 uncultured bacterium cyp153 W0UAI3 gamma proteobacterium HIMB55 OMB55_00002070 H3NWG4 Bradyrhizobium sp. DFCI-1 C207_06143 U1H776 gamma proteobacterium HIMB55 OMB55_00014510 H3NWP3 marine gamma proteobacterium HTCC2080 MGP2080_06587 A0Z166 Burkholderia xenovorans (strain LB400) Bx_A3593 Q143U3 Alcanivorax sp. P2S70 Q670_08165 U7G5C1 Marinobacter hydrocarbonoclasticus ATCC 49840 MARHY3773 H8WA08 Marinobacter sp. EVN1 Q672_10645 U7NYR4 uncultured bacterium P450 Q33DS4 uncultured bacterium cyp153 W0UDA1 uncultured bacterium cyp153 W0UCR5 uncultured bacterium cyp153 W0UD97 uncultured bacterium cyp153 W0UD81 uncultured bacterium cyp153 W0UCN3 uncultured bacterium cyp153 W0UCN5 uncultured bacterium cyp153 W0UCT3 gamma proteobacterium HdN1 ahpG HDN1F_17560 E1VKJ7 Marinobacter adhaerens (strain HP15) HP15_p187g148 E4PSB0 uncultured bacterium P450 Q33DT0 uncultured bacterium P450 Q33DS5 uncultured bacterium cyp153 W0UD61 uncultured bacterium P450 Q33DT1 Alcanivorax hongdengensis B3U002 uncultured bacterium P450 Q33DT2 uncultured bacterium P450 Q33DS7 uncultured bacterium cyp153 W0UCL9 uncultured bacterium cyp153 W0UDB3 Hyphomonas neptunium (strain ATCC 15444) HNE_2042 Q0C0K3 Alcanivorax dieselolei (strain DSM 16502/CGMCC ahpG B5T_02075 K0C9X8 1.3690/B-5) Alcanivorax hongdengensis A-11-3 A11A3_15327 L0WAH6 Alcanivorax dieselolei p450 D0Q1H3 Alcanivorax pacificus W11-5 S7S_02138 K2GI89 Marinobacter sp. ES-1 Q666_09590 U7G612 Limnobacter sp. MED105 LMED105_04587 A6GLB5 Marinobacter aquaeolei (strain ATCC 700491/DSM Maqu_0600 A1TY82 11845/VT8) (Marinobacter hydrocarbonoclasticus (strain DSM 11845)) Marinobacter sp. EVN1 Q672_13925 U7NUC4 Marinobacter sp. EN3 Q673_05250 U7H5S5 Marinobacter manganoxydans MnI7-9 KYE_03215 G6YPH4 Marinobacter hydrocarbonoclasticus ATCC 49840 ahpG2 MARHY2838 H8WCT8 Marinobacter hydrocarbonoclasticus (Pseudomonas ahpG2 MARHY2838 D9UAS2 nautica) Patulibacter medicamentivorans PAI11_40170 H0EAZ2 Acinetobacter baumannii WC-141 ACINWC141_2468 K8ZRD3 Saccharomonospora marina XMU15 SacmaDRAFT_5365 H5X733 Mycobacterium marinum (strain ATCC BAA-535/M) cyp153A16 MMAR_3154 B2HGN5 Mycobacterium abscessus 3A-0930-R p450 MA3A0930R_2169 I9I3J4 Mycobacterium abscessus 3A-0930-S p450 MA3A0930S_1729 I9I1F6 Mycobacterium abscessus 3A-0731 p450 MA3A0731_2042 I9GVU0 Mycobacterium abscessus 3A-0119-R p450 MA3A0119R_2080 I9FPY3 Mycobacterium abscessus 6G-0728-R p450 MA6G0728R_2104 I9DR77 Mycobacterium abscessus subsp. bolletii 1S-154-0310 p450 MM1S1540310_1492 I9CBZ2 Mycobacterium abscessus 6G-0728-S p450 MA6G0728S_5133 I9A485 Mycobacterium abscessus 3A-0810-R p450 MM3A0810R_2169 I8Q799 Mycobacterium abscessus 3A-0122-S p450 MA3A0122S_1691 I8LTR4 Mycobacterium abscessus 3A-0122-R p450 MA3A0122R_2136 I8L4A4 Mycobacterium abscessus 6G-0212 p450 MA6G0212_2171 I8I9K7 Mycobacterium abscessus subsp. bolletii 1S-153-0915 p450 MM1S1530915_1484 I8H4G3 Mycobacterium abscessus subsp. bolletii 1S-152-0914 p450 MM1S1520914_2142 I8GFC6 Mycobacterium abscessus subsp. bolletii 1S-151-0930 p450 MM1S1510930_1936 I8G1R8 Mycobacterium abscessus 6G-1108 p450 MA6G1108_2106 I8G118 Mycobacterium abscessus 6G-0125-S p450 MA6G0125S_2116 I8F2E6 Mycobacterium abscessus 6G-0125-R p450 MA6G0125R_1143 I8EZ93 Mycobacterium abscessus subsp. bolletii 2B-0307 p450 MM2B0307_1166 I9EQ97 Mycobacterium abscessus subsp. bolletii 2B-0107 p450 MM2B0107_1179 I8Q7R9 Mycobacterium abscessus subsp. bolletii 2B-1231 p450 MM2B1231_1908 I8PT86 Mycobacterium abscessus subsp. bolletii 2B-0912-S p450 MM2B0912S_1850 I8KHB7 Mycobacterium abscessus subsp. bolletii 2B-0912-R p450 MM2B0912R_2246 I8JU18 Mycobacterium abscessus subsp. bolletii 2B-0626 p450 MM2B0626_1842 I8HVB7 uncultured bacterium cyp153 W0UAF0 Parvibaculum lavamentivorans (strain DS-1/DSM 13023/ Plav_1951 A7HUI3 NCIMB 13966) Alcanivorax hongdengensis G1C7L3 Alcanivorax sp. DG881 ADG881_2119 B4X0H6 Marinobacter sp. C1S70 Q667_02605 U7NVJ0 marine gamma proteobacterium HTCC2143 GP2143_06784 A0YGW0 Alcanivorax sp. P2S70 Q670_00635 U7G5B3 Marinobacter goseongensis p450 T1WMH0 gamma proteobacterium BDW918 DOK_13444 I2JHG9 Hirschia baltica (strain ATCC 49814/DSM 5838/IFAM Hba1_0836 C6XQ13 1418) Acinetobacter indicus CIP 110367 P253_02820 V2UD76 Acinetobacter indicus ANC 4215 F956_01111 S3N495 Acinetobacter sp. OC4 cyp Q2MHE2 Acinetobacter baumannii NIPH 527 F921_03852 N9HTE2 Acinetobacter sp. CIP 102129 F973_00680 N8UI43 Acinetobacter sp. NIPH 809 F993_03507 N8P4U1 Acinetobacter baumannii OIFC0162 ACIN5162_A0021 K5DS46 Acinetobacter sp. EB104 nonM Q93SX3 Dietzia cinnamea P4 ES5_05410 E6J787 Acinetobacter sp. WC-743 ACINWC743_A0288 L9LSK8 Acinetobacter baumannii WC-348 ACINWC348_A0080 K9B8A0 Acinetobacter baumannii WC-141 ACINWC141_A0026 K8ZRU7 Acinetobacter baumannii WC-323 ACINWC323_A0095 K9AWS1 Gordonia malaquae NBRC 108250 GM1_050_00120 M3VCF1 Rhodococcus erythropolis SK121 RHOER0001_0266 C3JL15 Acinetobacter sp. COS3 Q674_03885 U7GP11 Acinetobacter guillouiae MSP4-18 L291_2817 S3YTQ7 Acinetobacter gyllenbergii MTCC 11365 L293_2966 S3YIH4 Acinetobacter gyllenbergii CIP 110306 F957_03919 S3MT86 Acinetobacter sp. CIP 110321 F896_03869 R9AJ00 Acinetobacter pittii ANC 3678 F930_03216 N9FYL9 Acinetobacter beijerinckii CIP 110307 F933_03106 N9FFM7 Acinetobacter beijerinckii CIP 110307 F933_03106 N9FFM7 Acinetobacter guillouiae CIP 63.46 F981_00071 N8TRF0 Acinetobacter sp. NIPH 236 F992_00196 N8PQM8 Acinetobacter radioresistens DSM 6976 = NBRC 102413 = ACRAD_64_00110 K6W366 CIP 103788 F939_02890 Acinetobacter sp. NBRC 100985 ACT4_067_00170 G7GIJ8 Williamsia sp. D3 W823_14840 V8CZP3 Rhodococcus ruber BKS 20-38 G352_16177 M2XNX0 Gordonia neofelifaecis NRRL B-59395 SCNU_19987 F1YPY6 Nocardioidaceae bacterium Broad-1 NBCG_04744 E9V105 Rhodococcus erythropolis DN1 N601_30795 T5HW62 Rhodococcus erythropolis (strain PR4/NBRC 100887) RER_pREL1-02600 Q3L9B0 Rhodococcus erythropolis DN1 N601_30930 T5HZQ2 Alcanivorax dieselolei B0LCZ6 uncultured bacterium cyp153 W0UD28 uncultured bacterium cyp153 W0UD53 uncultured bacterium cyp153 W0UCL1 Alcanivorax borkumensis ahpG1 Q5K134 Alcanivorax sp. 97CO-5 Y017_09710 W6ZMW5 Alcanivorax borkumensis (strain SK2/ATCC 700651/ p450 ABO_0201 ABO_2288 Q0VM62 DSM 11573) Alcanivorax borkumensis ahpG2 Q5K133 gamma proteobacterium HIMB55 OMB55_00008700 H3NSZ4 Amycolicicoccus subflavus (strain DSM 45089/DQS3-9A1) AS9A_4287 F6EL57 Dietzia cinnamea P4 ES5_17094 E6JDU2 Rhodococcus sp. R04 G0YY52 Dietzia sp. DQ12-45-1b L7QFU8 Gordonia terrae C-6 GTC6_22847 R7Y2Z3 Gordonia rubripertincta NBRC 101908 GORBP_030_00030 L7K246 Gordonia polyisoprenivorans NBRC 16320 GOPIP_035_00030 H0RD32 Gordonia amicalis NBRC 100051 = JCM 11271 GOAMI_32_00650 L7L3E0 Nocardia cyriacigeorgica (strain GUH-2) NOCYR_1539 H6R8V9 Mycobacterium gilvum (strain PYR-GCK) Mflv_4592 A4TFM0 (Mycobacterium flavescens (strain ATCC 700033/PYR-GCK)) Acinetobacter sp. ANC 3862 F900_00467 N9M6H3 Rhodococcus erythropolis (strain PR4/NBRC 100887) RER_pREL1-02830 Q3L987 Mycobacterium rhodesiae (strain NBB3) MycrhN_5185 G8RXP7 Rhodococcus wratislaviensis IFP 2016 Rwratislav_02222 L2TWM7 Nocardioides sp. CF8 CF8_1774 R7XZ06 Rhodococcus sp. AW25M09 RHODMAR_4781 L8DQ69 Mycobacterium sp. (strain MCS) Mmcs_3218 Q1B709 Mycobacterium sp. (strain JLS) Mjls_3229 A3Q1I0 Mycobacterium sp. (strain KMS) Mkms_3280 A1UI16 Mycobacterium intracellulare MOTT-02 OCO_23030 H8J7G3 Mycobacterium abscessus subsp. bolletii str. GO 06 linC MYCMA_1074 I6ZDN8 Mycobacterium abscessus (strain ATCC 19977/DSM MAB_2048c B1MP79 44196) Mycobacterium abscessus V06705 M879_18655 T0B128 Mycobacterium abscessus M94 S7W_02670 I0PWL5 Mycobacterium avium subsp. hominissuis 10-4249 O971_10910 V7M646 Mycobacterium parascrofulaceum ATCC BAA-614 HMPREF0591_1257 D5P513 Rhodococcus sp. AW25M09 RHODMAR_0629 L8DBR6 Nocardia asteroides NBRC 15531 NCAST_16_00270 U5E995 Aeromicrobium marinum DSM 15272 HMPREF0063_10876 E2SA86 Mycobacterium abscessus MAB_091912_2446 L833_0535 V6ZS84 Mycobacterium abscessus MAB_082312_2258 L830_0536 V6ZGT1 Mycobacterium abscessus 47J26 MAB47J26_13072 G6X6X6 Nocardioides sp. CF8 CF8_1685 R7XZ92 Gordonia polyisoprenivorans NBRC 16320 GOPIP_007_00470 H0R8L5 Gordonia araii NBRC 100433 GOARA_078_00570 G7H6Y3 marine gamma proteobacterium HTCC2080 MGP2080_13483 A0Z5X9 Gordonia paraffinivorans NBRC 108238 GP2_063_00030 M3V7L0 Planctomyces maris DSM 8797 PM8797T_18726 A6CH25 Amycolicicoccus subflavus (strain DSM 45089/DQS3-9A1) AS9A_2813 F6EJ28 Candidatus Microthrix parvicella RN1 BN381_420018 R4Z0X4 Gordonia paraffinivorans NBRC 108238 GP2_036_00650 M3TVA1 Nocardioides sp. CF8 CF8_2601 R7XVN2 Mycobacterium chubuense (strain NBB4) Mycch_5830 D2K2F1 Gordonia polyisoprenivorans (strain DSM 44266/VH2) GPOL_c44990 H6MXH6 Aeromicrobium marinum DSM 15272 HMPREF0063_10264 E2S8A7 Gordonia rubripertincta NBRC 101908 GORBP_109_00410 L7KEM4 Gordonia namibiensis NBRC 108229 GONAM_02_01570 K6XIG5 Gordonia sp. KTR9 KTR9_5380 J9STN3 Gordonia terrae NBRC 100016 GOTRE_050_00060 H5UDF7 Gordonia alkanivorans NBRC 16433 GOALK_030_00300 F9VS44 Gordonia alkanivorans goaBAC B3IX64 Gordonia sp. TF6 aoxA A9CMS7 Alcanivorax borkumensis (strain SK2/ATCC 700651/ Q6RCE3 DSM 11573) Gordonia malaquae NBRC 108250 GM1_011_00750 M3UVQ9 alpha proteobacterium JLT2015 C725_0051 M2TQQ4 Oceanicola batsensis HTCC2597 OB2597_05915 A3TT18 Sphingobium baderi LL03 L485_17855 T0HGM2 Erythrobacter litoralis (strain HTCC2594) ELI_12445 Q2N6W0 Erythrobacter sp. SD-21 ED21_18817 A5P986 Novosphingobium nitrogenifigens DSM 19370 Y88_2850 F1Z4F0 Sphingopyxis macrogoltabida (Sphingomonas ahpG5 Q5F4D8 macrogoltabidus) Sphingopyxis alaskensis (strain DSM 13593/LMG 18877/ Sala_2865 Q1GP52 RB2256) (Sphingomonas alaskensis) Sphingopyxis macrogoltabida (Sphingomonas ahpG4 Q5F4D1 macrogoltabidus) Novosphingobium aromaticivorans (strain DSM 12444) Saro_0220 Q2GBV5 Dickeya dadantii (strain Ech586) Dd586_1369 D2BW78 Sphingopyxis sp. MC1 EBMC1_05939 N9UVB0 Dietzia sp. D5 W0C650 Sphingobium indicum B90A SIDU_06697 I5BFE4 Sphingobium chinhatense IP26 M527_09955 W1KG42 Sphingobium sp. HDIP04 L286_21540 T0G3B9 Erythrobacter sp. NAP1 NAP1_13673 A3WFL2 Dickeya dadantii (strain 3937) (Erwinia chrysanthemi Dda3937_03358 E0SIQ2 (strain 3937)) Sphingomonas sanxanigenens DSM 19645 = NX02 NX02_10200 W0AB84 Sphingopyxis sp. MC1 EBMC1_03994 N9WE44 Dickeya sp. D s0432-1 A544_2711 U6Z9W7 Novosphingobium aromaticivorans (strain DSM 12444) Saro_1821 Q2G7B2 Erythrobacter litoralis (strain HTCC2594) ELI_09815 Q2N8D6 Parvibaculum lavamentivorans (strain DS-1/DSM 13023/ Plav_0029 A7HP19 NCIMB 13966) Novosphingobium pentaromativorans US6-1 NSU_3817 G6EHJ6

In some embodiments, the invention provide methods for synthesizing olefinic alcohol products as described above, wherein the enzyme is selected from AlkB, AlkB P1, and AlkB1 AB. In some embodiments, the enzyme is selected from CYP153 M. sp.; CYP153A M. aq.; CYP153A M. aq. (G307A); Cyp153A M. aq. (G307A)-CPR_(BM3); Cyp153A P.sp.-CPR_(BM3); CYP153A13N2; CYP153A13N3; CYP153A13P2; and CYP153A7. In some embodiments, the enzyme is selected from CYP52A13 and CYP52A3.

In a related aspect, the invention provides a whole cell catalyst comprising an enzyme capable of selectively hydroxylating one terminal carbon of an unsaturated or saturated hydrocarbon substrate. In some embodiments, the cell is a microbial cell. In some embodiments, the enzyme is selected from the group consisting of a non-heme diiron monooxygenase, a long-chain alkane hydroxylase, a cytochrome P450, and combinations thereof. In some embodiments, the enzyme is selected from Table 3, Table 4, Table 5, or a variant thereof having at least 90% identity thereto.

TABLE 6 Exemplary strains suitable for the present invention. Species Plasmid Genotype (or relevant gene deletions) Reference E. coli K12 GEc137 pGEc47J contains alkBFGKL alkST Grant et al. Enzyme Microb. Technol. 2011 E. coli W3110 pBT10 contains alkBFG alkST Schrewe et al. Adv. Synth. Cat. 2011 E. coli W3110 pBTL10 contains alkBFGL alkST Julsing et al. Adv. Synth. Cat. 2011 E. coli BL21(DE3) pET-28a(+)-LadA contains LadA Dong el al. Appl. Microbiol. Biotechnol. 2012 E. coli BL21(DE3) pET-28a(+)-CYP153A6 operon Gudimichi et al. Appl. Microbiol. Biotechnol. 2012 E. coli JM109 pJOE-CYP153A_(M. aq.)(G307A)-CPR_(BM3) Scheps et al. Microb. Biotechnol. 2013 E. coli HMS174 pET-28(+)-CYP153A_(M. aq.)(G307A)-CPR_(BM3) E. coli HMS174 pColaDuet-1-CYP153A_(M. aq.)(G307A)-CPR_(BM3), alkL E. coli HMS174 pET-28(+)-CYP153A_(P. sp.)-CPR_(BM3) Malca et al. Chem. Comm. 2012 C. tropicalis DP522 DP1 Δcyp52a17/Δ cyp52a18 Δ Lu et al. J. Am. Chem. cyp52a13/Δcyp52a14 Δ fao1/Δ fao1b Δ fao2a/Δ Soc. 2010 fao2b Δcyp52a12/Δcyp52a12b Δadh-a4/Δadh-a4b Δadhb4/Δadh-b4b Δadh-a10 Δ adh-b11 pXICL::CYP52A13 C. tropicalis DP526 DP1 Δcyp52a17/Δ cyp52a18 Δ cyp52a13/Δcyp52a14 Δ fao1/Δ fao1b Δ fao2a/Δ fao2b Δcyp52a12/Δcyp52a12b Δadh-a4/Δadh-a4b Δadhb4/Δadh-b4b Δadh-a10 Δ adh-b11 pXICL::CYP52A12 C. tropicalis DP428 DP1 Δcyp52a17/Δ cyp52a18 Δ cyp52a13/Δcyp52a14 Δ fao1/Δ fao1b Δ fao2a/Δ fao2b Δcyp52a12/Δcyp52a12b Δadh-a4/Δadh-a4b Δadhb4/Δadh-b4b Δadh-a10 Δ adh-b11 pXICL::CYP52A17

The methods of the invention allow for the production of terminal alcohols with controlled regioselectivity, while disfavoring the formation of unwanted species such as epoxides or elimination products. The stereochemistry of an olefinic alcohol product will depend on factors including the structure of the particular olefinic substrate used in a particular reaction, as well as the identity of the enzyme. The methods of the invention can be conducted with enzymes that are selective for particular substrates (e.g., cis or Z alkenes vs. trans or E alkenes), as well as with enzymes that demonstrate terminal selectivity (e.g., hydroxylation of one end of an asymmetric alkene vs. the other end of the asymmetric alkene).

In certain instances, a hydroxylase enzyme will exhibit catalytic efficiency with one isomer of an internal alkene (e.g., the cis or Z isomer of an internal alkene) that is greater than the catalytic efficiency exhibited with the other isomer of the same internal alkene (e.g., the trans or E isomer of an internal alkene). In some embodiments, the invention provides methods wherein the catalytic efficiency of the hydroxylase enzyme is at least about 2-fold greater with one isomer of an internal alkene than with the other isomer of the internal alkene. The catalytic efficiency exhibited by a hydroxylase with one isomer of an internal alkene can be, for example, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, or at least about 500-fold greater than the catalytic efficiency exhibited by the hydroxylase with the other isomer of the internal alkene.

A particular enzyme can therefore produce Z product over E product from a mixture of Z and E isomeric substrates or enrich the Z product over the E product. In certain embodiments, the invention provides methods for preparing olefinic alcohol products wherein the Z:E (cis:trans) isomeric ratio of the olefinic alcohol product is different from the Z:E (cis:trans) isomeric ratio of the olefinic substrate. The Z:E isomeric ratio of the olefinic alcohol product can be, for example, around 2 times greater than the Z:E isomeric ratio of the olefinic substrate. The Z:E isomeric ratio of the olefinic alcohol product can be, for example, around 1.25 times, 1.5 times, 2 times, 2.5 times, 3 times, 4 times, 5 times, 10 times, 20 times, 30 times, or 40 times greater than the Z:E isomeric ratio of the olefinic substrate.

In some embodiments, the invention provides methods for preparing olefinic alcohol products wherein the E:Z (trans:cis) isomeric ratio of the olefinic alcohol product is different from the E:Z (trans:cis) isomeric ratio of the olefinic substrate. The E:Z isomeric ratio of the olefinic alcohol product can be, for example, around 2 times greater than the E:Z isomeric ratio of the olefinic substrate. The E:Z isomeric ratio of the olefinic alcohol product can be, for example, around 1.25 times, 1.5 times, 2 times, 2.5 times, 3 times, 4 times, 5 times, 10 times, 20 times, 30 times, or 40 times greater than the E:Z isomeric ratio of the olefinic substrate.

In some embodiments, the Z:E isomeric ratio of the olefinic alcohol is about 1.25 times greater than the Z:E isomeric ratio of the olefinic substrate. In some embodiments, the E:Z isomeric ratio of the olefinic alcohol is about 1.25 times greater than the E:Z isomeric ratio of the olefinic substrate.

In certain instances, the biohydroxylation reactions in the methods of the invention have the potential to form a mixture of two or more products from the same substrate. When an olefinic substrate is asymmetric, for example, hydroxylation of one end/terminus of the substrate leads to one product while hydroxylation of the other end/terminus of the substrate leads to a different product. A reaction could therefore result in a mixture of two olefinic alcohol products. The terminal isomer ratio of an asymmetric olefinic alcohol product can range from about 1:99 to about 99:1. The terminal isomer ratio can be, for example, from about 1:99 to about 1:75, or from about 1:75 to about 1:50, or from about 1:50 to about 1:25, or from about 99:1 to about 75:1, or from about 75:1 to about 50:1, or from about 50:1 to about 25:1. The terminal isomer ratio can be from about 1:80 to about 1:20, or from about 1:60 to about 1:40, or from about 80:1 to about 20:1 or from about 60:1 to about 40:1. The terminal isomer ratio can be about 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, or about 1:95. The terminal isomer ratio can be about 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, or about 95:1.

The distribution of a product mixture can be expressed as a regioselectivity percentage (“regioselectivity %”). Taking the reaction in FIG. 3 as a non-limiting example, for instance, the regioselectivity of (Z)-5-hexadecene hydroxylation can be calculated using the formula: regioselectivity %=[(χ₁₁)/(χ₁₁+χ₅)]×100%, wherein χ₁₁ is the mole fraction for (Z)-11-hexadecen-1-ol and wherein χ₅ is the mole fraction for (Z)-5-hexadecen-1-ol. In general, the regioselectivity % with respect to terminal alcohol isomers ranges from about 1% to about 99%. The regioselectivity % can be from about 1% to about 99%, or from about 20% to about 80%, or from about 40% to about 60%, or from about 1% to about 25%, or from about 25% to about 50%, or from about 50% to about 75%. The regioselectivity % can be at least about about 5% 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

In some embodiments, the regioselectivity % is at least about 60%. In some embodiments, the regioselectivity % is at least about 60% and the Z:E isomeric ratio of the olefinic alcohol is about 1.25 times greater than the Z:E isomeric ratio of the olefinic substrate.

In certain instances, varying levels of olefin epoxidation will occur during the biohydroxylation reactions used in the methods of the invention. See, e.g., Scheme 7. Epoxidation of terminal alkenes, in particular, can occur when certain hydroxylase enzymes are used. It is often desirable to minimize such epoxidation or avoid the formation of epoxides altogether. Typically, methods of the invention are conducted with hydroxylase enzymes that produce product mixtures with alcohol product:epoxide ratios of at least 1:1. The alcohol product:epoxide ratio can range from about 1:1 to about 99:1. The alcohol:epoxide ratio can be, for example, from about 99:1 to about 75:1, or from about 75:1 to about 50:1, or from about 50:1 to about 25:1. The alcohol:epoxide ratio can be from about 80:1 to about 20:1 or from about 60:1 to about 40:1. The alcohol:epoxide ratio can be about 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, or about 95:1.

In some embodiments, methods are conducted using an enzyme that produces an olefinic alcohol product:epoxide product ratio of greater than 1:1. In some embodiments, the enzyme produces an olefinic alcohol product: epoxide product ratio of greater than 2:1.

The distribution of a product mixture can be expressed as a percent selectivity for hydroxylation vs. epoxidation. Taking the reaction in Scheme 7a as a non-limiting example, the percent selectivity for hydroxylation vs. epoxidation of a terminal alkene can be calculated using the formula: selectivity %=[(χ_(H))/(χ_(H)+χ_(E))]×100%, wherein χ_(H) is the mole fraction for the hydroxylation product (i.e., the terminal olefinic alcohol) and wherein χ_(E) is the mole fraction for the epoxidation product (i.e., the terminal epoxide). In general, the percent selectivity for hydroxylation vs. epoxidation ranges from about 1% to about 99%. The percent selectivity for hydroxylation vs. epoxidation can be from about 1% to about 99%, or from about 20% to about 80%, or from about 40% to about 60%, or from about 1% to about 25%, or from about 25% to about 50%, or from about 50% to about 75%. The percent selectivity for hydroxylation vs. epoxidation can be about 5% 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

When halogen-substituted substrates are used in the methods of the invention, varying levels of dehalogenation can occur during hydroxylation. Dehalogenation typically results in the formation of aldehyde byproduct. Preferably, dehalogenation is minimized or avoided during the hydroxylation reactions. Typically, methods of the invention are conducted with hydroxylase enzymes that produce product mixtures with alcohol:aldehyde ratios of at least 1:1. The alcohol:aldehyde ratio of the product can range from about 1:1 to about 99:1. The alcohol:aldehyde ratio can be, for example, from about 99:1 to about 75:1, or from about 75:1 to about 50:1, or from about 50:1 to about 25:1. The alcohol:aldehyde ratio can be from about 80:1 to about 20:1 or from about 60:1 to about 40:1. The alcohol:aldehyde ratio can be about 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, or about 95:1.

The distribution of a product mixture can be expressed as a percent selectivity for hydroxylation vs. dehalogenation. The percent selectivity for hydroxylation vs. dehalogenation of a halogen-substituted substrate can be calculated using the formula: selectivity %=[(χ_(H))/(χ_(H)+χ_(A))]×100%, wherein χ_(H) is the mole fraction for the hydroxylation product and wherein χ_(A) is the mole fraction for the aldehyde product. In general, the percent selectivity for hydroxylation vs. dehalogenation ranges from about 1% to about 99%. The percent selectivity for hydroxylation vs. dehalogenation can be from about 1% to about 99%, or from about 20% to about 80%, or from about 40% to about 60%, or from about 1% to about 25%, or from about 25% to about 50%, or from about 50% to about 75%. The percent selectivity for hydroxylation vs. dehalogenation can be about 5% 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

Synthesis of Terminal Alkenals

As indicated above, the alcohol moiety generated via hydroxylation can be further modified to generate alkenals or acetate esters.

Oxidation of Fatty Alcohols

Oxidation of fatty alcohols is often achieved via selective oxidation via pyridinium chlorocrhomate (PCC) (Scheme 20).

Alternatively, TEMPO (TEMPO=2,2,6,6-tetramethylpiperidinyl-N-oxyl) and related catalyst systems can be used to selectively oxidize alcohols to aldehydes. These methods are described in Ryland and Stahl (2014), herein incorporated by reference in its entirety.

Bio-oxidation of Terminal Alcohols

Many insect pheromones are fatty aldehydes or comprise a fatty aldehyde component. As such, the conversion of the fatty alcohol produced via terminal hydroxylation to the fatty aldehyde is required to produce certain pheromones. The conversion of a fatty alcohol to a fatty aldehyde is known to be catalyzed by alcohol dehydrogenases (ADH) and alcohol oxidases (AOX). Additionally, the conversion of a length C_(n) fatty acid to a C_(n-1) fatty aldehyde is catalyzed by plant α-dioxygenases (α-DOX) (Scheme 21).

The present invention describes enzymes that oxidize fatty alcohols to fatty aldehydes.

In some embodiments, an alcohol oxidase (AOX) is used to catalyze the conversion of a fatty alcohol to a fatty aldehyde. Alcohol oxidases catalyze the conversion of alcohols into corresponding aldehydes (or ketones) with electron transfer via the use of molecular oxygen to form hydrogen peroxide as a by-product. AOX enzymes utilize flavin adenine dinucleotide (FAD) as an essential cofactor and regenerate with the help of oxygen in the reaction medium. Catalase enzymes may be coupled with the AOX to avoid accumulation of the hydrogen peroxide via catalytic conversion into water and oxygen.

Based on the substrate specificities, AOXs may be categorized into four groups: (a) short chain alcohol oxidase, (b) long chain alcohol oxidase, (c) aromatic alcohol oxidase, and (d) secondary alcohol oxidase (Goswami et al. 2013). Depending on the chain length of the desired substrate, some member of these four groups are better suited than others as candidates for evaluation.

Short chain alcohol oxidases (including but not limited to those currently classified as EC 1.1.3.13, Table 7) catalyze the oxidation of lower chain length alcohol substrates in the range of C1-C8 carbons (van der Klei et al. 1991) (Ozimek et al. 2005). Aliphatic alcohol oxidases from methylotrophic yeasts such as Candida boidinii and Komagataella pastoris (formerly Pichia pastoris) catalyze the oxidation of primary alkanols to the corresponding aldehydes with a preference for unbranched short-chain aliphatic alcohols. The most broad substrate specificity is found for alcohol oxidase from the Pichia pastoris including propargyl alcohol, 2-chloroethanol, 2-cyanoethanol (Dienys et al. 2003). The major challenge encountered in alcohol oxidation is the high reactivity of the aldehyde product. Utilization of a two liquid phase system (water/solvent) can provide in-situ removal of the aldehyde product from the reaction phase before it is further converted to the acid. For example, hexanal production from hexanol using Pichia pastoris alcohol oxidase coupled with bovine liver catalase was achieved in a bi-phasic system by taking advantage of the presence of a stable alcohol oxidase in aqueous phase (Karra-Chaabouni et al. 2003). For example, alcohol oxidase from Pichia pastoris was able to oxidize aliphatic alcohols of C6 to C11 when used biphasic organic reaction system (Murray and Duff 1990). Methods for using alcohol oxidases in a biphasic system according to (Karra-Chaabouni et al. 2003) and (Murray and Duff 1990) are incorporated by reference in their entirety.

Long chain alcohol oxidases (including but not limited to those currently classified as EC 1.1.3.20; Table 8) include fatty alcohol oxidases, long chain fatty acid oxidases, and long chain fatty alcohol oxidases that oxidize alcohol substrates with carbon chain length of greater than six (Goswami et al. 2013). Banthorpe et al. reported a long chain alcohol oxidase purified from the leaves of Tanacetum vulgare that was able to oxidize saturated and unsaturated long chain alcohol substrates including hex-trans-2-en-1-ol and octan-1-ol (Banthorpe 1976) (Cardemil 1978). Other plant species, including Simmondsia chinensis (Moreau, R. A., Huang 1979), Arabidopsis thaliana (Cheng et al. 2004), and Lotus japonicas (Zhao et al. 2008) have also been reported as sources of long chain alcohol oxidases. Fatty alcohol oxidases are mostly reported from yeast species (Hommel and Ratledge 1990) (Vanhanen et al. 2000) (Hommel et al. 1994) (Kemp et al. 1990) and these enzymes play an important role in long chain fatty acid metabolism (Cheng et al. 2005). Fatty alcohol oxidases from yeast species that degrade and grow on long chain alkanes and fatty acid catalyze the oxidation of fatty alcohols. Fatty alcohol oxidase from Candida tropicalis has been isolated as microsomal cell fractions and characterized for a range of substrates (Eirich et al. 2004) (Kemp et al. 1988) (Kemp et al. 1991) (Mauersberger et al. 1992). Significant activity is observed for primary alcohols of length C₈ to C₁₆ with reported K_(M) in the 10-50 μM range (Eirich et al. 2004). Alcohol oxidases described may be used for the conversion of medium chain aliphatic alcohols to aldehydes as described, for example, for whole-cells Candida boidinii (Gabelman and Luzio 1997), and Pichia pastoris (Duff and Murray 1988) (Murray and Duff 1990). Long chain alcohol oxidases from filamentous fungi were produced during growth on hydrocarbon substrates (Kumar and Goswami 2006) (Savitha and Ratledge 1991). The long chain fatty alcohol oxidase (LjFAO1) from Lotus japonicas has been heterologously expressed in E. coli and exhibited broad substrate specificity for alcohol oxidation including 1-dodecanol and 1-hexadecanol (Zhao et al. 2008).

TABLE 7 Alcohol oxidase enzymes capable of oxidizing short chain alcohols (EC 1.1.3.13) Organism Gene names Accession No. Komagataella pastoris (strain ATCC 76273/CBS 7435/ AOX1 PP7435_Chr4-0130 F2QY27 CECT 11047/NRRL Y-11430/Wegner 21-1) (Yeast) (Pichia pastoris) Komagataella pastoris (strain GS115/ATCC 20864) AOX1 PAS_chr4_0821 P04842 (Yeast) (Pichia pastoris) Komagataella pastoris (strain ATCC 76273/CBS 7435/ AOX2 PP7435_Chr4-0863 F2R038 CECT 11047/NRRL Y-11430/Wegner 21-1) (Yeast) (Pichia pastoris) Komagataella pastoris (strain GS115/ATCC 20864) AOX2 PAS_chr4_0152 C4R702 (Yeast) (Pichia pastoris) Candida boidinii (Yeast) AOD1 Q00922 Pichia angusta (Yeast) (Hansenula polymorpha) MOX P04841 Thanatephorus cucumeris (strain AG1-IB/isolate AOD1 BN14_10802 M5CC52 7/3/14) (Lettuce bottom rot fungus) (Rhizoctonia solani) Thanatephorus cucumeris (strain AG1-IB/isolate MOX BN14_12214 M5CF32 7/3/14) (Lettuce bottom rot fungus) (Rhizoctonia solani) Thanatephorus cucumeris (strain AG1-IB/isolate AOD1 BN14_10691 M5CAV1 7/3/14) (Lettuce bottom rot fungus) (Rhizoctonia solani) Thanatephorus cucumeris (strain AG1-IB/isolate AOD1 BN14_09479 M5C7F4 7/3/14) (Lettuce bottom rot fungus) (Rhizoctonia solani) Thanatephorus cucumeris (strain AG1-IB/isolate AOD1 BN14_10803 M5CB66 7/3/14) (Lettuce bottom rot fungus) (Rhizoctonia solani) Thanatephorus cucumeris (strain AG1-IB/isolate AOD1 BN14_09900 M5C9N9 7/3/14) (Lettuce bottom rot fungus) (Rhizoctonia solani) Thanatephorus cucumeris (strain AG1-IB/isolate AOD1 BN14_08302 M5C2L8 7/3/14) (Lettuce bottom rot fungus) (Rhizoctonia solani) Thanatephorus cucumeris (strain AG1-IB/isolate MOX BN14_09408 M5C784 7/3/14) (Lettuce bottom rot fungus) (Rhizoctonia solani) Thanatephorus cucumeris (strain AG1-IB/isolate MOX BN14_09478 M5C8F8 7/3/14) (Lettuce bottom rot fungus) (Rhizoctonia solani) Thanatephorus cucumeris (strain AG1-IB/isolate AOD1 BN14_11356 M5CH40 7/3/14) (Lettuce bottom rot fungus) (Rhizoctonia solani) Ogataea henricii AOD1 A5LGF0 Candida methanosorbosa AOD1 A5LGE5 Candida methanolovescens AOD1 A5LGE4 Candida succiphila AOD1 A5LGE6 Aspergillus niger (strain CBS 513.88/FGSC A1513) An15g02200 A2R501 Aspergillus niger (strain CBS 513.88/FGSC A1513) An18g05480 A2RB46 Moniliophthora perniciosa (Witches'-broom disease I7CMK2 fungus) (Marasmius perniciosus) Candida cariosilignicola AOD1 A5LGE3 Candida pignaliae AOD1 A5LGE1 Candida pignaliae AOD2 A5LGE2 Candida sonorensis AOD1 A5LGD9 Candida sonorensis AOD2 A5LGE0 Pichia naganishii AOD1 A5LGF2 Ogataea minuta AOD1 A5LGF1 Ogataea philodendri AOD1 A5LGF3 Ogataea wickerhamii AOD1 A5LGE8 Kuraishia capsulata AOD1 A5LGE7 Talaromyces stipitatus (strain ATCC 10500/CBS TSTA_021940 B8MHF8 375.48/QM 6759/NRRL 1006) (Penicillium stipitatum) Talaromyces stipitatus (strain ATCC 10500/CBS TSTA_065150 B8LTH7 375.48/QM 6759/NRRL 1006) (Penicillium stipitatum) Talaromyces stipitatus (strain ATCC 10500/CBS TSTA_065150 B8LTH8 375.48/QM 6759/NRRL 1006) (Penicillium stipitatum) Talaromyces stipitatus (strain ATCC 10500/CBS TSTA_000410 B8MSB1 375.48/QM 6759/NRRL 1006) (Penicillium stipitatum) Ogataea glucozyma AOD1 A5LGE9 Ogataea parapolymorpha (strain DL-1/ATCC 26012/ HPODL_03886 W1QCJ3 NRRL Y-7560) (Yeast) (Hansenula polymorpha) Gloeophyllum trabeum (Brown rot fungus) AOX A8DPS4 Pichia angusta (Yeast) (Hansenula polymorpha) mox1 A6PZG8 Pichia trehalophila AOD1 A5LGF4 Pichia angusta (Yeast) (Hansenula polymorpha) mox1 A6PZG9 Pichia angusta (Yeast) (Hansenula polymorpha) mox1 A6PZG7 Ixodes scapularis (Black-legged tick) (Deer tick) IscW_ISCW017898 B7PIZ7

TABLE 8 Alcohol oxidase enzymes capable of oxidizing long chain alcohols including fatty alcohols (EC 1.1.3.20) Organism Gene names Accession No. Lotus japonicus (Lotus corniculatus var. japonicus) FAO1 B5WWZ8 Arabidopsis thaliana (Mouse-ear cress) FAO1 At1g03990 F21M11.7 Q9ZWB9 Lotus japonicus (Lotus corniculatus var. japonicus) FAO2 B5WWZ9 Arabidopsis thaliana (Mouse-ear cress) FAO3 At3g23410 MLM24.14 Q9LW56 MLM24.23 Arabidopsis thaliana (Mouse-ear cress) FAO4A At4g19380 T5K18.160 O65709 Arabidopsis thaliana (Mouse-ear cress) FAO4B At4g28570 T5F17.20 Q94BP3 Microbotryum violaceum (strain p1A1 Lamole) MVLG_06864 U5HIL4 (Anther smut fungus) (Ustilago violacea) Ajellomyces dermatitidis ATCC 26199 BDFG_03507 T5BNQ0 Gibberella zeae (strain PH-1/ATCC MYA-4620/ FG06918.1 FGSG_06918 I1RS14 FGSC 9075/NRRL 31084) (Wheat head blight fungus) (Fusarium graminearum) Pichia sorbitophila (strain ATCC MYA-4447/ Piso0_004410 G8Y5E1 BCRC 22081/CBS 7064/NBRC 10061/NRRL GNLVRS01_PISO0K16268g Y-12695) (Hybrid yeast) GNLVRS01_PISO0L16269g Emericella nidulans (strain FGSC A4/ATCC AN0623.2 ANIA_00623 Q5BFQ7 38163/CBS 112.46/NRRL 194/M139) (Aspergillus nidulans) Pyrenophora tritici-repentis (strain Pt-1C-BFP) PTRG_10154 B2WJW5 (Wheat tan spot fungus) (Drechslera tritici-repentis) Paracoccidioides lutzii (strain ATCC MYA-826/ PAAG_09117 C1HEC6 Pb01) (Paracoccidioides brasiliensis) Candida parapsilosis (strain CDC 317/ATCC CPAR2_204420 G8BG15 MYA-4646) (Yeast) (Monilia parapsilosis) Pseudozyma brasiliensis (strain GHG001) (Yeast) PSEUBRA_SCAF2g03010 V5GPS6 Candida parapsilosis (strain CDC 317/ATCC CPAR2_204430 G8BG16 MYA-4646) (Yeast) (Monilia parapsilosis) Sclerotinia borealis F-4157 SBOR_5750 W9CDE2 Sordaria macrospora (strain ATCC MYA-333/ SMAC_06361 F7W6K4 DSM 997/K(L3346)/K-hell) Sordaria macrospora (strain ATCC MYA-333/ SMAC_01933 F7VSA1 DSM 997/K(L3346)/K-hell) Meyerozyma guilliermondii (strain ATCC 6260/ PGUG_03467 A5DJL6 CBS 566/DSM 6381/JCM 1539/NBRC 10279/ NRRL Y-324) (Yeast) (Candida guilliermondii) Trichophyton rubrum CBS 202.88 H107_00669 A0A023ATC5 Arthrobotrys oligospora (strain ATCC 24927/CBS AOL_s00097g516 G1XJI9 115.81/DSM 1491) (Nematode-trapping fungus) (Didymozoophaga oligospora) Scheffersomyces stipitis (strain ATCC 58785/CBS FAO1 PICST_90828 A3LYX9 6054/NBRC 10063/NRRL Y-11545) (Yeast) (Pichia stipitis) Scheffersomyces stipitis (strain ATCC 58785/CBS FAO2 PICST_32359 A3LW61 6054/NBRC 10063/NRRL Y-11545) (Yeast) (Pichia stipitis) Aspergillus oryzae (strain 3.042) (Yellow koji mold) Ao3042_09114 I8TL25 Fusarium oxysporum (strain Fo5176) (Fusarium FOXB_17532 F9GFU8 vascular wilt) Rhizopus delemar (strain RA 99-880/ATCC MYA-4621/ RO3G_08271 I1C536 FGSC 9543/NRRL 43880) (Mucormycosis agent) (Rhizopus arrhizus var. delemar) Rhizopus delemar (strain RA 99-880/ATCC MYA-4621/ RO3G_00154 I1BGX0 FGSC 9543/NRRL 43880) (Mucormycosis agent) (Rhizopus arrhizus var. delemar) Fusarium oxysporum (strain Fo5176) (Fusarium FOXB_07532 F9FMA2 vascular wilt) Penicillium roqueforti PROQFM164_S02g001772 W6QPY1 Aspergillus clavatus (strain ATCC 1007/CBS ACLA_018400 A1CNB5 513.65/DSM 816/NCTC 3887/NRRL 1) Arthroderma otae (strain ATCC MYA-4605/CBS MCYG_08732 C5G1B0 113480) (Microsporum canis) Trichophyton tonsurans (strain CBS 112818) (Scalp TESG_07214 F2S8I2 ringworm fungus) Colletotrichum higginsianum (strain IMI 349063) CH063_13441 H1VUE7 (Crucifer anthracnose fungus) Ajellomyces capsulatus (strain H143) (Darling's HCDG_07658 C6HN77 disease fungus) (Histoplasma capsulatum) Trichophyton rubrum (strain ATCC MYA-4607/ TERG_08235 F2T096 CBS 118892) (Athlete's foot fungus) Cochliobolus heterostrophus (strain C5/ATCC COCHEDRAFT_1201414 M2UMT9 48332/race O) (Southern corn leaf blight fungus) (Bipolaris maydis) Candida orthopsilosis (strain 90-125) (Yeast) CORT_0D04510 H8X643 Candida orthopsilosis (strain 90-125) (Yeast) CORT_0D04520 H8X644 Candida orthopsilosis (strain 90-125) (Yeast) CORT_0D04530 H8X645 Pseudozyma aphidis DSM 70725 PaG_03027 W3VP49 Coccidioides posadasii (strain C735) (Valley fever CPC735_000380 C5P005 fungus) Magnaporthe oryzae (strain P131) (Rice blast OOW_P131scaffold01214g15 L7IZ92 fungus) (Pyricularia oryzae) Neurospora tetrasperma (strain FGSC 2508/ATCC NEUTE1DRAFT_82541 F8MKD1 MYA-4615/P0657) Hypocrea virens (strain Gv29-8/FGSC 10586) TRIVIDRAFT_54537 G9MMY7 (Gliocladium virens) (Trichoderma virens) Hypocrea virens (strain Gv29-8/FGSC 10586) TRIVIDRAFT_53801 G9MT89 (Gliocladium virens) (Trichoderma virens) Aspergillus niger (strain CBS 513.88/FGSC An01g09620 A2Q9Z3 A1513) Verticillium dahliae (strain VdLs.17/ATCC MYA-4575/ VDAG_05780 G2X6J8 FGSC 10137) (Verticillium wilt) Ustilago maydis (strain 521/FGSC 9021) (Corn UM02023.1 Q4PCZ0 smut fungus) Fusarium oxysporum f. sp. lycopersici MN25 FOWG_13006 W9LNI9 Fusarium oxysporum f. sp. lycopersici MN25 FOWG_02542 W9N9Z1 Candida tropicalis (Yeast) FAO1 Q6QIR6 Magnaporthe oryzae (strain 70-15/ATCC MYA-4617/ MGG_11317 G4MVK1 FGSC 8958) (Rice blast fungus) (Pyricularia oryzae) Candida tropicalis (Yeast) faot Q9P8D9 Candida tropicalis (Yeast) FAO2a Q6QIR5 Phaeosphaeria nodorum (strain SN15/ATCC SNOG_02371 Q0V0U3 MYA-4574/FGSC 10173) (Glume blotch fungus) (Septoria nodorum) Candida tropicalis (Yeast) FAO2b Q6QIR4 Pestalotiopsis fici W106-1 PFICI_11209 W3WU04 Magnaporthe oryzae (strain Y34) (Rice blast OOU_Y34scaffold00240g57 L7IFT5 fungus) (Pyricularia oryzae) Pseudogymnoascus destructans (strain ATCC GMDG_01756 L8G0G6 MYA-4855/20631-21) (Bat white-nose syndrome fungus) (Geomyces destructans) Pseudogymnoascus destructans (strain ATCC GMDG_04950 L8GCY2 MYA-4855/20631-21) (Bat white-nose syndrome fungus) (Geomyces destructans) Mycosphaerella fijiensis (strain CIRAD86) (Black MYCFIDRAFT_52380 M2Z831 leaf streak disease fungus) (Pseudocercospora fijiensis) Bipolaris oryzae ATCC 44560 COCMIDRAFT_84580 W7A0I8 Cladophialophora psammophila CBS 110553 A1O5_08147 W9WTM9 Fusarium oxysporum f. sp. melonis 26406 FOMG_05173 X0AEE6 Fusarium oxysporum f. sp. melonis 26406 FOMG_17829 W9ZBB7 Cyphellophora europaea CBS 101466 HMPREF1541_02174 W2S2S5 Aspergillus kawachii (strain NBRC 4308) (White AKAW_00147 G7X626 koji mold) (Aspergillus awamori var. kawachi) Aspergillus terreus (strain NIH 2624/FGSC ATEG_05086 Q0CMJ8 A1156) Coccidioides immitis (strain RS) (Valley fever CIMG_02987 J3KAI8 fungus) Ajellomyces dermatitidis (strain ER-3/ATCC BDCG_04701 C5GLS5 MYA-2586) (Blastomyces dermatitidis) Fusarium oxysporum f. sp. cubense (strain race 1) FOC1_g10013865 N4U732 (Panama disease fungus) Rhodotorula glutinis (strain ATCC 204091/IIP 30/ RTG_00643 G0SVU8 MTCC 1151) (Yeast) Aspergillus niger (strain ATCC 1015/CBS 113.46/ ASPNIDRAFT_35778 G3XTM6 FGSC A1144/LSHB Ac4/NCTC 3858a/NRRL 328/USDA 3528.7) Candida cloacae fao1 Q9P8D8 Candida cloacae fao2 Q9P8D7 Fusarium oxysporum f. sp. cubense (strain race 1) FOC1_g10006358 N4TUH3 (Panama disease fungus) Candida albicans (strain SC5314/ATCC MYA-2876) FAO1 CaO19.13562 Q59RS8 (Yeast) orf19.13562 Candida albicans (strain SC5314/ATCC MYA-2876) FAO1 CaO19.6143 orf19.6143 Q59RP0 (Yeast) Chaetomium thermophilum (strain DSM 1495/ CTHT_0018560 G0S2U9 CBS 144.50/IMI 039719) Mucor circinelloides f. circinelloides (strain HMPREF1544_05296 S2JDN0 1006PhL) (Mucormycosis agent) (Calyptromyces circinelloides) Mucor circinelloides f. circinelloides (strain HMPREF1544_05295 S2JYP5 1006PhL) (Mucormycosis agent) (Calyptromyces circinelloides) Mucor circinelloides f. circinelloides (strain HMPREF1544_06348 S2JVK9 1006PhL) (Mucormycosis agent) (Calyptromyces circinelloides) Botryotinia fuckeliana (strain BcDW1) (Noble rot BcDW1_6807 M7UD26 fungus) (Botrytis cinerea) Podospora anserina (strain S/ATCC MYA-4624/ PODANS_5_13040 B2AFD8 DSM 980/FGSC 10383) (Pleurage anserina) Neosartorya fumigata (strain ATCC MYA-4609/ AFUA_1G17110 Q4WR91 Af293/CBS 101355/FGSC A1100) (Aspergillus fumigatus) Fusarium oxysporum f. sp. vasinfectum 25433 FOTG_00686 X0MEE6 Fusarium oxysporum f. sp. vasinfectum 25433 FOTG_12485 X0LE98 Trichophyton interdigitale H6 H101_06625 A0A022U717 Beauveria bassiana (strain ARSEF 2860) (White BBA_04100 J4UNY3 muscardine disease fungus) (Tritirachium shiotae) Fusarium oxysporum f. sp. radicis-lycopersici 26381 FOCG_00843 X0GQ62 Fusarium oxysporum f. sp. radicis-lycopersici 26381 FOCG_15170 X0F4T1 Neurospora tetrasperma (strain FGSC 2509/P0656) NEUTE2DRAFT_88670 G4UNN6 Pseudozyma hubeiensis (strain SY62) (Yeast) PHSY_000086 R9NVU1 Lodderomyces elongisporus (strain ATCC 11503/ LELG_03289 A5E102 CBS 2605/JCM 1781/NBRC 1676/NRRL YB-4239) (Yeast) (Saccharomyces elongisporus) Malassezia globosa (strain ATCC MYA-4612/CBS MGL_3855 A8QAY8 7966) (Dandruff-associated fungus) Byssochlamys spectabilis (strain No. 5/NBRC PVAR5_7014 V5GBL6 109023) (Paecilomyces variotii) Ajellomyces capsulatus (strain H88) (Darling's HCEG_03274 F0UF47 disease fungus) (Histoplasma capsulatum) Trichosporon asahii var. asahii (strain ATCC 90039/ A1Q1_03669 J6FBP4 CBS 2479/JCM 2466/KCTC 7840/NCYC 2677/ UAMH 7654) (Yeast) Penicillium oxalicum (strain 114-2/CGMCC 5302) PDE_00027 S7Z8U8 (Penicillium decumbens) Fusarium oxysporum f. sp. conglutinans race 2 FOPG_02304 X0IBE3 54008 Fusarium oxysporum f. sp. conglutinans race 2 FOPG_13066 X0H540 54008 Fusarium oxysporum f. sp. raphani 54005 FOQG_00704 X0D1G8 Fusarium oxysporum f. sp. raphani 54005 FOQG_10402 X0C482 Metarhizium acridum (strain CQMa 102) MAC_03115 E9DZR7 Arthroderma benhamiae (strain ATCC MYA-4681/ ARB_02250 D4B1C1 CBS 112371) (Trichophyton mentagrophytes) Fusarium oxysporum f. sp. cubense tropical race 4 FOIG_12161 X0JFI6 54006 Fusarium oxysporum f. sp. cubense tropical race 4 FOIG_12751 X0JDU5 54006 Cochliobolus heterostrophus (strain C4/ATCC COCC4DRAFT_52836 N4WZZ0 48331/race T) (Southern corn leaf blight fungus) (Bipolaris maydis) Trichosporon asahii var. asahii (strain CBS 8904) A1Q2_00631 K1VZW1 (Yeast) Mycosphaerella graminicola (strain CBS 115943/ MYCGRDRAFT_37086 F9X375 IPO323) (Speckled leaf blotch fungus) (Septoria tritici) Botryotinia fuckeliana (strain T4) (Noble rot fungus) BofuT4_P072020.1 G2XQ18 (Botrytis cinerea) Metarhizium anisopliae (strain ARSEF 23/ATCC MAA_05783 E9F0I4 MYA-3075) Cladophialophora carrionii CBS 160.54 G647_05801 V9DAR1 Coccidioides posadasii (strain RMSCC 757/ CPSG_09174 E9DH75 Silveira) (Valley fever fungus) Rhodosporidium toruloides (strain NP11) (Yeast) RHTO_06879 M7X159 (Rhodotorula gracilis) Puccinia graminis f. sp. tritici (strain CRL PGTG_10521 E3KIL8 75-36-700-3/race SCCL) (Black stem rust fungus) Trichophyton rubrum CBS 288.86 H103_00624 A0A022WG28 Colletotrichum fioriniae PJ7 CFIO01_08202 A0A010RKZ4 Trichophyton rubrum CBS 289.86 H104_00611 A0A022XB46 Cladophialophora yegresii CBS 114405 A1O7_02579 W9WC55 Colletotrichum orbiculare (strain 104-T/ATCC Cob_10151 N4VFP3 96160/CBS 514.97/LARS 414/MAFF 240422) (Cucumber anthracnose fungus) (Colletotrichum lagenarium) Drechslerella stenobrocha 248 DRE_03459 W7IDL6 Neosartorya fumigata (strain CEA10/CBS 144.89/ AFUB_016500 B0XP90 FGSC A1163) (Aspergillus fumigatus) Thielavia terrestris (strain ATCC 38088/NRRL THITE_2117674 G2R8H9 8126) (Acremonium alabamense) Gibberella fujikuroi (strain CBS 195.34/IMI 58289/ FFUJ_02948 S0DZP7 NRRL A-6831) (Bakanae and foot rot disease fungus) (Fusarium fujikuroi) Gibberella fujikuroi (strain CBS 195.34/IMI 58289/ FFUJ_ 12030 S0EMC6 NRRL A-6831) (Bakanae and foot rot disease fungus) (Fusarium fujikuroi) Aspergillus flavus (strain ATCC 200026/FGSC AFLA_109870 B8N941 A1120/NRRL 3357/JCM 12722/SRRC 167) Togninia minima (strain UCR-PA7) (Esca disease UCRPA7_1719 R8BTZ6 fungus) (Phaeoacremonium aleophilum) Ajellomyces dermatitidis (strain ATCC 18188/ BDDG_09783 F2TUC0 CBS 674.68) (Blastomyces dermatitidis) Macrophomina phaseolina (strain MS6) (Charcoal MPH_10582 K2RHA5 rot fungus) Neurospora crassa (strain ATCC 24698/74-OR23-1A/ NCU08977 Q7S2Z2 CBS 708.71/DSM 1257/FGSC 987) Neosartorya fischeri (strain ATCC 1020/DSM NFIA_008260 A1D156 3700/FGSC A1164/NRRL 181) (Aspergillus fischerianus) Fusarium pseudograminearum (strain CS3096) FPSE_11742 K3U9J5 (Wheat and barley crown-rot fungus) Spathaspora passalidarum (strain NRRL Y-27907/ SPAPADRAFT_54193 G3AJP0 11-Y1) Spathaspora passalidarum (strain NRRL Y-27907/ SPAPADRAFT_67198 G3ANX7 11-Y1) Trichophyton verrucosum (strain HKI 0517) TRV_07960 D4DL86 Arthroderma gypseum (strain ATCC MYA-4604/ MGYG_07264 E4V2J0 CBS 118893) (Microsporum gypseum) Hypocrea jecorina (strain QM6a) (Trichoderma TRIREDRAFT_43893 G0R7P8 reesei) Trichophyton rubrum MR1448 H110_00629 A0A022Z1G4 Aspergillus ruber CBS 135680 EURHEDRAFT_512125 A0A017SPR0 Glarea lozoyensis (strain ATCC 20868/MF5171) GLAREA_04397 S3D6C1 Setosphaeria turcica (strain 28A) (Northern leaf SETTUDRAFT_20639 R0K6H8 blight fungus) (Exserohilum turcicum) Paracoccidioides brasiliensis (strain Pb18) PADG_06552 C1GH16 Fusarium oxysporum Fo47 FOZG_13577 W9JPG9 Fusarium oxysporum Fo47 FOZG_05344 W9KPH3 Trichophyton rubrum MR1459 H113_00628 A0A022ZY09 Penicillium marneffei (strain ATCC 18224/CBS PMAA_075740 B6QBY3 334.59/QM 7333) Sphaerulina musiva (strain SO2202) (Poplar stem SEPMUDRAFT_154026 M3DAK6 canker fungus) (Septoria musiva) Gibberella moniliformis (strain M3125/FGSC FVEG_10526 W7N4P8 7600) (Maize ear and stalk rot fungus) (Fusarium verticillioides) Gibberella moniliformis (strain M3125/FGSC FVEG_08281 W7MVR9 7600) (Maize ear and stalk rot fungus) (Fusarium verticillioides) Pseudozyma antarctica (strain T-34) (Yeast) PANT_22d00298 M9MGF2 (Candida antarctica) Paracoccidioides brasiliensis (strain Pb03) PABG_07795 C0SJD4 Rhizophagus irregularis (strain DAOM 181602/ GLOINDRAFT_82554 U9TF61 DAOM 197198/MUCL 43194) (Arbuscular mycorrhizal fungus) (Glomus intraradices) Penicillium chrysogenum (strain ATCC 28089/ Pc21g23700 B6HJ58 DSM 1075/Wisconsin 54-1255) (Penicillium PCH_Pc21g23700 notatum) Baudoinia compniacensis (strain UAMH 10762) BAUCODRAFT_274597 M2M6Z5 (Angels' share fungus) Hypocrea atroviridis (strain ATCC 20476/IMI TRIATDRAFT_280929 G9NJ32 206040) (Trichoderma atroviride) Colletotrichum gloeosporioides (strain Cg-14) CGLO_06642 T0LPH0 (Anthracnose fungus) (Glomerella cingulata) Cordyceps militaris (strain CM01) (Caterpillar CCM_02665 G3JB34 fungus) Pyronema omphalodes (strain CBS 100304) PCON_13062 U4LKE9 (Pyronema confluens) Colletotrichum graminicola (strain M1.001/M2/ GLRG_08499 E3QR67 FGSC 10212) (Maize anthracnose fungus) (Glomerella graminicola) Glarea lozoyensis (strain ATCC 74030/MF5533) M7I_2117 H0EHX4 Fusarium oxysporum f. sp. cubense (strain race 4) FOC4_g10002493 N1S969 (Panama disease fungus) Fusarium oxysporum f. sp. cubense (strain race 4) FOC4_g10011461 N1RT80 (Panama disease fungus) Cochliobolus sativus (strain ND90Pr/ATCC COCSADRAFT_295770 M2TBE4 201652) (Common root rot and spot blotch fungus) (Bipolaris sorokiniana) Mixia osmundae (strain CBS 9802/IAM 14324/ Mo05571 E5Q_05571 G7E7S3 JCM 22182/KY 12970) Mycosphaerella pini (strain NZE10/CBS 128990) DOTSEDRAFT_69651 N1PXR0 (Red band needle blight fungus) (Dothistroma septosporum) Grosmannia clavigera (strain kw1407/UAMH CMQ_1113 F0XC64 11150) (Blue stain fungus) (Graphiocladiella clavigera) Fusarium oxysporum FOSC 3-a FOYG_03004 W9IUE5 Fusarium oxysporum FOSC 3-a FOYG_16040 W9HNP0 Fusarium oxysporum FOSC 3-a FOYG_17058 W9HB31 Nectria haematococca (strain 77-13-4/ATCC NECHADRAFT_37686 C7YQL1 MYA-4622/FGSC 9596/MPVI) (Fusarium solani subsp. pisi) Nectria haematococca (strain 77-13-4/ATCC NECHADRAFT_77262 C7ZJI0 MYA-4622/FGSC 9596/MPVI) (Fusarium solani subsp. pisi) Tuber melanosporum (strain Mel28) (Perigord black GSTUM_00010376001 D5GLS0 truffle) Ajellomyces dermatitidis (strain SLH14081) BDBG_07633 C5JYI9 (Blastomyces dermatitidis) Chaetomium globosum (strain ATCC 6205/CBS CHGG_09885 Q2GQ69 148.51/DSM 1962/NBRC 6347/NRRL 1970) (Soil fungus) Candida tenuis (strain ATCC 10573/BCRC 21748/ CANTEDRAFT_108652 G3B9Z1 CBS 615/JCM 9827/NBRC 10315/NRRL Y-1498/ VKM Y-70) (Yeast) Trichophyton rubrum CBS 100081 H102_00622 A0A022VKY4 Pyrenophora teres f. teres (strain 0-1) (Barley net PTT_09421 E3RLZ3 blotch fungus) (Drechslera teres f. teres) Colletotrichum gloeosporioides (strain Nara gc5) CGGC5_4608 L2GB29 (Anthracnose fungus) (Glomerella cingulata) Gibberella zeae (Wheat head blight fungus) FG05_06918 A0A016PCS4 (Fusarium graminearum) Trichophyton soudanense CBS 452.61 H105_00612 A0A022Y6A6 Sclerotinia sclerotiorum (strain ATCC 18683/1980/ SS1G_07437 A7EQ37 Ss-1) (White mold) (Whetzelinia sclerotiorum) Fusarium oxysporum f. sp. pisi HDV247 FOVG_14401 W9NWU8 Fusarium oxysporum f. sp. pisi HDV247 FOVG_02874 W9Q5V3 Ustilago hordei (strain Uh4875-4) (Barley covered UHOR_03009 I2G1Z4 smut fungus) Sporisorium reilianum (strain SRZ2) (Maize head sr12985 E6ZYF7 smut fungus) Bipolaris zeicola 26-R-13 COCCADRAFT_81154 W6YIP8 Melampsora larici-populina (strain 98AG31/ MELLADRAFT_78490 F4RUZ8 pathotype 3-4-7) (Poplar leaf rust fungus) Fusarium oxysporum f. sp. lycopersici (strain 4287/ FOXG_01901 J9MG95 CBS 123668/FGSC 9935/NRRL 34936) (Fusarium vascular wilt of tomato) Fusarium oxysporum f. sp. lycopersici (strain 4287/ FOXG_11941 J9N9S4 CBS 123668/FGSC 9935/NRRL 34936) (Fusarium vascular wilt of tomato) Bipolaris victoriae FI3 COCVIDRAFT_39053 W7EMJ8 Debaryomyces hansenii (strain ATCC 36239/CBS DEHA2E04268g Q6BQL4 767/JCM 1990/NBRC 0083/IGC 2968) (Yeast) (Torulaspora hansenii) Clavispora lusitaniae (strain ATCC 42720) (Yeast) CLUG_01505 C4XZX3 (Candida lusitaniae) Candida albicans (strain WO-1) (Yeast) CAWG_02023 C4YME4 Trichophyton rubrum MR850 H100_00625 A0A022U0Q2 Candida dubliniensis (strain CD36/ATCC MYA-646/ CD36_32890 B9WMC7 CBS 7987/NCPF 3949/NRRL Y-17841) (Yeast) Starmerella bombicola AOX1 A0A024FB95 Thielavia heterothallica (strain ATCC 42464/ MYCTH_103590 G2QJL7 BCRC 31852/DSM 1799) (Myceliophthora thermophila) Claviceps purpurea (strain 20.1) (Ergot fungus) CPUR_07614 M1WFI4 (Sphacelia segetum) Aspergillus oryzae (strain ATCC 42149/RIB 40) AO090023000571 Q2UH61 (Yellow koji mold) Dictyostelium discoideum (Slime mold) DDB_0184181 Q54DT6 DDB_G0292042 Triticum urartu (Red wild einkorn) (Crithodium TRIUR3_22733 M7YME5 urartu) Solanum tuberosum (Potato) PGSC0003DMG400017211 M1BG07 Oryza sativa subsp. japonica (Rice) OSJNBb0044B19.5 Q8W5P8 LOC_Os10g33540 Oryza sativa subsp. japonica (Rice) OJ1234_B11.20 Q6K9N5 Os02g0621800 Oryza sativa subsp. japonica (Rice) OSJNBa0001K12.5 Q8W5P3 LOC_Os10g33520 Zea mays (Maize) ZEAMMB73_809149 C0P3J6 Citrus clementina CICLE_v10011111mg V4S9P4 Citrus clementina CICLE_v10018992mg V4U4C9 Citrus clementina CICLE_v10004405mg V4S9D3 Citrus clementina CICLE_v10004403mg V4RZZ6 Morus notabilis L484_011703 W9RIK0 Morus notabilis L484_005930 W9RET7 Medicago truncatula (Barrel medic) (Medicago MTR_1g075650 G7I4U3 tribuloides) Arabidopsis thaliana (Mouse-ear cress) Q8LDP0 Medicago truncatula (Barrel medic) (Medicago MTR_4g081080 G7JF07 tribuloides) Simmondsia chinensis (Jojoba) (Buxus chinensis) L7VFV2 Prunus persica (Peach) (Amygdalus persica) PRUPE_ppa018458mg M5VXL1 Aphanomyces astaci H257_07411 W4GI89 Aphanomyces astaci H257_07412 W4GI44 Aphanomyces astaci H257_07411 W4GKE3 Aphanomyces astaci H257_07411 W4GK29 Aphanomyces astaci H257_07411 W4GJ79 Aphanomyces astaci H257_07411 W4GI38 Phaeodactylum tricornutum (strain CCAP 1055/1) PHATRDRAFT_48204 B7G6C1 Hordeum vulgare var. distichum (Two-rowed barley) F2E4R4 Hordeum vulgare var. distichum (Two-rowed barley) F2DZG1 Hordeum vulgare var. distichum (Two-rowed barley) M0YPG7 Hordeum vulgare var. distichum (Two-rowed barley) M0YPG6 Hordeum vulgare var. distichum (Two-rowed barley) F2CUY4 Ricinus communis (Castor bean) RCOM_0867830 B9S1S3 Brassica rapa subsp. pekinensis (Chinese cabbage) BRA014947 M4DEM5 (Brassica pekinensis) Ricinus communis (Castor bean) RCOM_0258730 B9SV13 Brassica rapa subsp. pekinensis (Chinese cabbage) BRA001912 M4CCI2 (Brassica pekinensis) Brassica rapa subsp. pekinensis (Chinese cabbage) BRA012548 M4D7T8 (Brassica pekinensis) Brassica rapa subsp. pekinensis (Chinese cabbage) BRA024190 M4E5Y6 (Brassica pekinensis) Brassica rapa subsp. pekinensis (Chinese cabbage) BRA015283 M4DFL0 (Brassica pekinensis) Ricinus communis (Castor bean) RCOM_1168730 B9SS54 Zea mays (Maize) C4J691 Oryza glaberrima (African rice) I1P2B7 Zea mays (Maize) B6SXM3 Zea mays (Maize) C0HFU4 Aegilops tauschii (Tausch's goatgrass) (Aegilops F775_19577 R7W4J3 squarrosa) Solanum habrochaites (Wild tomato) (Lycopersicon R9R6T0 hirsutum) Physcomitrella patens subsp. patens (Moss) PHYPADRAFT_124285 A9S535 Physcomitrella patens subsp. patens (Moss) PHYPADRAFT_113581 A9RG13 Physcomitrella patens subsp. patens (Moss) PHYPADRAFT_182504 A9S9A5 Solanum pennellii (Tomato) (Lycopersicon R9R6Q1 pennellii) Vitis vinifera (Grape) VIT_02s0087g00630 F6HJ27 Vitis vinifera (Grape) VIT_07s0005g03780 F6HZM3 Vitis vinifera (Grape) VIT_05s0049g01400 F6H8T4 Vitis vinifera (Grape) VITISV_019349 A5AH38 Capsella rubella CARUB_v10013046mg R0HIT3 Capsella rubella CARUB_v10004212mg R0GUX4 Capsella rubella CARUB_v10004208mg R0F3X6 Capsella rubella CARUB_v10012453mg R0ILD0 Capsella rubella CARUB_v10004208mg R0GUX1 Eutrema salsugineum (Saltwater cress) EUTSA_v10024496mg V4MD54 (Sisymbrium salsugineum) Eutrema salsugineum (Saltwater cress) EUTSA_v10020141mg V4NM59 (Sisymbrium salsugineum) Eutrema salsugineum (Saltwater cress) EUTSA_v10024496mg V4LUR9 (Sisymbrium salsugineum) Eutrema salsugineum (Saltwater cress) EUTSA_v10024528mg V4P767 (Sisymbrium salsugineum) Eutrema salsugineum (Saltwater cress) EUTSA_v10006882mg V4L2P6 (Sisymbrium salsugineum) Selaginella moellendorffii (Spikemoss) SELMODRAFT_87684 D8R6Z6 Selaginella moellendorffii (Spikemoss) SELMODRAFT_87621 D8R6Z5 Selaginella moellendorffii (Spikemoss) SELMODRAFT_74601 D8QN81 Selaginella moellendorffii (Spikemoss) SELMODRAFT_73531 D8QN82 Sorghum bicolor (Sorghum) (Sorghum vulgare) Sb04g026390 C5XXS4 SORBIDRAFT_04g026390 Sorghum bicolor (Sorghum) (Sorghum vulgare) Sb04g026370 C5XXS1 SORBIDRAFT_04g026370 Sorghum bicolor (Sorghum) (Sorghum vulgare) Sb01g019470 C5WYH6 SORBIDRAFT_01g019470 Sorghum bicolor (Sorghum) (Sorghum vulgare) Sb01g019480 C5WYH7 SORBIDRAFT_01g019480 Sorghum bicolor (Sorghum) (Sorghum vulgare) Sb01g019460 C5WYH5 SORBIDRAFT_01g019460 Solanum pimpinellifolium (Currant tomato) R9R6J2 (Lycopersicon pimpinellifolium) Phaseolus vulgaris (Kidney bean) (French bean) PHAVU_007G124200g V7BGM7 Phaseolus vulgaris (Kidney bean) (French bean) PHAVU_011G136600g V7AI35 Phaseolus vulgaris (Kidney bean) (French bean) PHAVU_001G162800g V7D063 Solanum tuberosum (Potato) PGSC0003DMG400024294 M1C923 Solanum tuberosum (Potato) PGSC0003DMG400018458 M1BKV4 Solanum tuberosum (Potato) PGSC0003DMG400018458 M1BKV3 Glycine max (Soybean) (Glycine hispida) K7LK61 Glycine max (Soybean) (Glycine hispida) K7KXQ9 Populus trichocarpa (Western balsam poplar) POPTR_0008s16920g B9HKS3 (Populus balsamifera subsp. trichocarpa) Picea sitchensis (Sitka spruce) (Pinus sitchensis) B8LQ84 Populus trichocarpa (Western balsam poplar) POPTR_0004s24310g U5GKQ5 (Populus balsamifera subsp. trichocarpa) Populus trichocarpa (Western balsam poplar) POPTR_0010s07980g B9HSG9 (Populus balsamifera subsp. trichocarpa) Glycine max (Soybean) (Glycine hispida) I1N9S7 Glycine max (Soybean) (Glycine hispida) I1LSK5 Setaria italica (Foxtail millet) (Panicum italicum) Si034362m.g K4A658 Solanum lycopersicum (Tomato) (Lycopersicon Solyc09g072610.2 K4CUT7 esculentum) Setaria italica (Foxtail millet) (Panicum italicum) Si016380m.g K3YQ38 Solanum lycopersicum (Tomato) (Lycopersicon R9R6I9 esculentum) Solanum lycopersicum (Tomato) (Lycopersicon Solyc09g090350.2 K4CW61 esculentum) Solanum lycopersicum (Tomato) (Lycopersicon Solyc08g005630.2 K4CI54 esculentum) Solanum lycopersicum (Tomato) (Lycopersicon Solyc08g075240.2 K4CMP1 esculentum) Setaria italica (Foxtail millet) (Panicum italicum) Si034359m.g K4A655 Setaria italica (Foxtail millet) (Panicum italicum) Si034354m.g K4A650 Mimulus guttatus (Spotted monkey flower) (Yellow MIMGU_mgv1a001896mg A0A022PU07 monkey flower) Mimulus guttatus (Spotted monkey flower) (Yellow MIMGU_mgv1a022390mg A0A022RAV4 monkey flower) Mimulus guttatus (Spotted monkey flower) (Yellow MIMGU_mgv1a001868mg A0A022S2E6 monkey flower) Mimulus guttatus (Spotted monkey flower) (Yellow MIMGU_mgv1a001883mg A0A022S275 monkey flower) Mimulus guttatus (Spotted monkey flower) (Yellow MIMGU_mgv1a001761mg A0A022QNF0 monkey flower) Musa acuminata subsp. malaccensis (Wild banana) M0SNA8 (Musa malaccensis) Musa acuminata subsp. malaccensis (Wild banana) M0RUT7 (Musa malaccensis) Musa acuminata subsp. malaccensis (Wild banana) M0RUK3 (Musa malaccensis) Saprolegnia diclina VS20 SDRG_10901 T0RG89 Brachypodium distachyon (Purple false brome) BRADI3G49085 I1IBP7 (Trachynia distachya) Brachypodium distachyon (Purple false brome) BRADI3G28677 I1I4N2 (Trachynia distachya) Brachypodium distachyon (Purple false brome) BRADI3G28657 I1I4N0 (Trachynia distachya) Oryza sativa subsp. indica (Rice) OsI_34012 B8BHG0 Oryza sativa subsp. indica (Rice) OsI_08118 B8AFT8 Oryza sativa subsp. indica (Rice) OsI_34008 A2Z8H1 Oryza sativa subsp. indica (Rice) OsI_34014 B8BHG1 Oryza sativa subsp. japonica (Rice) LOC_Os10g33460 Q7XDG3 Oryza sativa subsp. japonica (Rice) Os10g0474800 Q0IX12 Oryza sativa subsp. japonica (Rice) Os10g0474966 C7J7R1 Oryza sativa subsp. japonica (Rice) OSJNBa0001K12.13 Q8W5N7 Oryza sativa subsp. japonica (Rice) OsJ_31873 B9G683 Oryza sativa subsp. japonica (Rice) OsJ_31875 B9G684 Oryza sativa subsp. japonica (Rice) OSJNBa0001K12.3 Q8W5P5 Arabidopsis lyrata subsp. lyrata (Lyre-leaved rock- ARALYDRAFT_470376 D7KDA3 cress) Arabidopsis lyrata subsp. lyrata (Lyre-leaved rock- ARALYDRAFT_479855 D7L3B6 cress) Arabidopsis lyrata subsp. lyrata (Lyre-leaved rock- ARALYDRAFT_491906 D7MDA9 cress) Arabidopsis lyrata subsp. lyrata (Lyre-leaved rock- ARALYDRAFT_914728 D7MGS9 cress)

In some embodiments, an alcohol dehydrogenase (ADH, Table 9) is used to catalyze the conversion of a fatty alcohol to a fatty aldehyde. A number of ADHs identified from alkanotrophic organisms, Pseudomonas fluorescens NRRL B-1244 (Hou et al. 1983), Pseudomonas butanovora ATCC 43655 (Vangnai and Arp 2001), and Acinetobacter sp. strain M-1 (Tani et al. 2000), have shown to be active on short to medium-chain alkyl alcohols (C₂ to C₁₄). Additionally, commercially available ADHs from Sigma, Horse liver ADH and Baker's yeast ADH have detectable activity for substrates with length C₁₀ and greater. The reported activities for the longer fatty alcohols may be impacted by the difficulties in solubilizing the substrates. For the yeast ADH from Sigma, little to no activity is observed for C₁₂ to C₁₄ aldehydes by (Tani et al. 2000), however, activity for C₁₂ and C₁₆ hydroxy-ω-fatty acids has been observed (Lu et al. 2010). Recently, two ADHs were characterized from Geobacillus thermodenitrificans NG80-2, an organism that degrades C₁₅ to C₃₆ alkanes using the LadA hydroxylase. Activity was detected from methanol to 1-triacontanol (C₃₀) for both ADHs, with 1-octanol being the preferred substrate for ADH2 and ethanol for ADH1 (Liu et al. 2009).

The use of ADHs in whole-cell bioconversions has been mostly focused on the production of chiral alcohols from ketones (Ernst et al. 2005) (Schroer et al. 2007). Using the ADH from Lactobacillus brevis and coupled cofactor regeneration with isopropanol, Schroer et al. reported the production of 797 g of (R)-methyl-3 hydroxybutanoate from methyl acetoacetate, with a space time yield of 29 g/L/h (Schroer et al. 2007). Examples of aliphatic alcohol oxidation in whole-cell transformations have been reported with commercially obtained S. cerevisiae for the conversion of hexanol to hexanal (Presecki et al. 2012) and 2-heptanol to 2-heptanone (Cappaert and Larroche 2004).

TABLE 9 Exemplary alcohol dehydrogenase enzymes. Organisms Gene Name Accession No. Bactrocera oleae (Olive fruit fly) (Dacus oleae) ADH Q9NAR7 Cupriavidus necator (Alcaligenes eutrophus) (Ralstonia adh P14940 eutropha) Drosophila adiastola (Fruit fly) (Idiomyia adiastola) Adh Q00669 Drosophila affinidisjuncta (Fruit fly) (Idiomyia Adh P21518 affinidisjuncta) Drosophila ambigua (Fruit fly) Adh P25139 Drosophila borealis (Fruit fly) Adh P48584 Drosophila differens (Fruit fly) Adh P22245 Drosophila equinoxialis (Fruit fly) Adh Q9NG42 Drosophila flavomontana (Fruit fly) Adh P48585 Drosophila guanche (Fruit fly) Adh Q09009 Drosophila hawaiiensis (Fruit fly) Adh P51549 Drosophila heteroneura (Fruit fly) Adh P21898 Drosophila immigrans (Fruit fly) Adh Q07588 Drosophila insularis (Fruit fly) Adh Q9NG40 Drosophila lebanonensis (Fruit fly) (Scaptodrosophila Adh P10807 lebanonensis) Drosophila mauritiana (Fruit fly) Adh P07162 Drosophila madeirensis (Fruit fly) Adh Q09010 Drosophila mimica (Fruit fly) (Idiomyia mimica) Adh Q00671 Drosophila nigra (Fruit fly) (Idiomyia nigra) Adh Q00672 Drosophila orena (Fruit fly) Adh P07159 Drosophila pseudoobscura bogotana (Fruit fly) Adh P84328 Drosophila picticornis (Fruit fly) (Idiomyia picticornis) Adh P23361 Drosophila planitibia (Fruit fly) Adh P23277 Drosophila paulistorum (Fruit fly) Adh Q9U8S9 Drosophila silvestris (Fruit fly) Adh P23278 Drosophila subobscura (Fruit fly) Adh Q03384 Drosophila teissieri (Fruit fly) Adh P28484 Drosophila tsacasi (Fruit fly) Adh P51550 Fragaria ananassa (Strawberry) ADH P17648 Malus domestica (Apple) (Pyres malus) ADH P48977 Scaptomyza albovittata (Fruit fly) Adh P25988 Scaptomyza crassifemur (Fruit fly) (Drosophila crassifemur) Adh Q00670 Sulfolobus sp. (strain RC3) adh P50381 Zaprionus tuberculatus (Vinegar fly) Adh P51552 Geobacillus stearothermophilus (Bacillus stearothermophilus) adh P42327 Drosophila mayaguana (Fruit fly) Adh, Adh2 P25721 Drosophila melanogaster (Fruit fly) Adh, CG3481 P00334 Drosophila pseudoobscura pseudoobscura (Fruit fly) Adh, GA17214 Q6LCE4 Drosophila simulans (Fruit fly) Adh, GD23968 Q24641 Drosophila yakuba (Fruit fly) Adh, GE19037 P26719 Drosophila ananassae (Fruit fly) Adh, GF14888 Q50L96 Drosophila erecta (Fruit fly) Adh, GG25120 P28483 Drosophila grimshawi (Fruit fly) (Idiomyia grimshawi) Adh, GH13025 P51551 Drosophila willistoni (Fruit fly) Adh, GK18290 Q05114 Drosophila persimilis (Fruit fly) Adh, GL25993 P37473 Drosophila sechellia (Fruit fly) Adh, GM15656 Q9GN94 Cupriavidus necator (strain ATCC 17699/H16/DSM 428/ adh, H16_A0757 Q0KDL6 Stanier 337) (Ralstonia eutropha) Mycobacterium tuberculosis (strain CDC 1551/Oshkosh) adh, MT1581 P9WQC2 Staphylococcus aureus (strain MW2) adh, MW0568 Q8NXU1 Mycobacterium tuberculosis (strain ATCC 25618/H37Rv) adh, Rv1530 P9WQC3 Staphylococcus aureus (strain N315) adh, SA0562 Q7A742 Staphylococcus aureus (strain bovine RF122/ET3-1) adh, SAB0557 Q2YSX0 Sulfolobus acidocaldarius (strain ATCC 33909/DSM 639/ adh, Saci_2057 Q4J781 JCM 8929/NBRC 15157/NCIMB 11770) Staphylococcus aureus (strain COL) adh, SACOL0660 Q5HI63 Staphylococcus aureus (strain NCTC 8325) adh, Q2G0G1 SAOUHSC_00608 Staphylococcus aureus (strain MRSA252) adh, SAR0613 Q6GJ63 Staphylococcus aureus (strain MSSA476) adh, SAS0573 Q6GBM4 Staphylococcus aureus (strain USA300) adh, SAUSA300_0594 Q2FJ31 Staphylococcus aureus (strain Mu50/ATCC 700699) adh, SAV0605 Q99W07 Staphylococcus epidermidis (strain ATCC 12228) adh, SE_0375 Q8CQ56 Staphylococcus epidermidis (strain ATCC 35984/RP62A) adh, SERP0257 Q5HRD6 Sulfolobus solfataricus (strain ATCC 35092/DSM 1617/ adh, SSO2536 P39462 JCM 11322/P2) Sulfolobus tokodaii (strain DSM 16993/JCM 10545/NBRC adh, STK_25770 Q96XE0 100140/7) Anas platyrhynchos (Domestic duck) (Anas boschas) ADH1 P30350 Apteryx australis (Brown kiwi) ADH1 P49645 Ceratitis capitata (Mediterranean fruit fly) (Tephritis capitata) ADH1 P48814 Ceratitis cosyra (Mango fruit fly) (Trypeta cosyra) ADH1 Q70UN9 Gallus gallus (Chicken) ADH1 P23991 Columba livia (Domestic pigeon) ADH1 P86883 Coturnix coturnix japonica (Japanese quail) (Coturnix ADH1 P19631 japonica) Drosophila hydei (Fruit fly) Adh1 P23236 Drosophila montana (Fruit fly) Adh1 P48586 Drosophila mettleri (Fruit fly) Adh1 P22246 Drosophila mulleri (Fruit fly) Adh1 P07161 Drosophila navojoa (Fruit fly) Adh1 P12854 Geomys attwateri (Attwater's pocket gopher) (Geomys ADH1 Q9Z2M2 bursarius attwateri) Geomys bursarius (Plains pocket gopher) ADH1 Q64413 Geomys knoxjonesi (Knox Jones's pocket gopher) ADH1 Q64415 Hordeum vulgare (Barley) ADH1 P05336 Kluyveromyces marxianus (Yeast) (Candida kefyr) ADH1 Q07288 Zea mays (Maize) ADH1 P00333 Mesocricetus auratus (Golden hamster) ADH1 P86885 Pennisetum americanum (Pearl millet) (Pennisetum glaucum) ADH1 P14219 Petunia hybrida (Petunia) ADH1 P25141 Oryctolagus cuniculus (Rabbit) ADH1 Q03505 Solanum tuberosum (Potato) ADH1 P14673 Struthio camelus (Ostrich) ADH1 P80338 Trifolium repens (Creeping white clover) ADH1 P13603 Zea luxurians (Guatemalan teosinte) (Euchlaena luxurians) ADH1 Q07264 Saccharomyces cerevisiae (strain ATCC 204508/S288c) ADH1, ADC1, P00330 (Baker's yeast) YOL086C, O0947 Arabidopsis thaliana (Mouse-ear cress) ADH1, ADH, P06525 At1g77120, F22K20.19 Schizosaccharomyces pombe (strain 972/ATCC 24843) adh1, adh, P00332 (Fission yeast) SPCC13B11.01 Drosophila lacicola (Fruit fly) Adh1, Adh-1 Q27404 Mus musculus (Mouse) Adh1, Adh-1 P00329 Peromyscus maniculatus (North American deer mouse) ADH1, ADH-1 P41680 Rattus norvegicus (Rat) Adh1, Adh-1 P06757 Drosophila virilis (Fruit fly) Adh1, Adh-1, B4M8Y0 GJ18208 Scheffersomyces stipitis (strain ATCC 58785/CBS 6054/ ADH1, ADH2, O00097 NBRC 10063/NRRL Y-11545) (Yeast) (Pichia stipitis) PICST_68558 Aspergillus flavus (strain ATCC 200026/FGSC A1120/ adh1, AFLA_048690 P41747 NRRL 3357/JCM 12722/SRRC 167) Neurospora crassa (strain ATCC 24698/74-OR23-1A/CBS adh-1, B17C10.210, Q9P6C8 708.71/DSM 1257/FGSC 987) NCU01754 Candida albicans (Yeast) ADH1, CAD P43067 Oryza sativa subsp. japonica (Rice) ADH1, DUPR11.3, Q2R8Z5 Os11g0210300, LOC_Os11g10480, OsJ_032001 Drosophila mojavensis (Fruit fly) Adh1, GI17644 P09370 Kluyveromyces lactis (strain ATCC 8585/CBS 2359/DSM ADH1, P20369 70799/NBRC 1267/NRRL Y-1140/WM37) (Yeast) KLLA0F21010g (Candida sphaerica) Oryza sativa subsp. indica (Rice) ADH1, OsI_034290 Q75ZX4 Pongo abelii (Sumatran orangutan) (Pongo pygmaeus abelii) ADH1A Q5RBP7 Homo sapiens (Human) ADH1A, ADH1 P07327 Macaca mulatta (Rhesus macaque) ADH1A, ADH1 P28469 Pan troglodytes (Chimpanzee) ADH1B Q5R1W2 Papio hamadryas (Hamadryas baboon) ADH1B P14139 Homo sapiens (Human) ADH1B, ADH2 P00325 Homo sapiens (Human) ADH1C, ADH3 P00326 Papio hamadryas (Hamadryas baboon) ADH1C, ADH3 O97959 Ceratitis capitata (Mediterranean fruit fly) (Tephritis capitata) ADH2 P48815 Ceratitis cosyra (Mango fruit fly) (Trypeta cosyra) ADH2 Q70UP5 Ceratitis rosa (Natal fruit fly) (Pterandrus rosa) ADH2 Q70UP6 Drosophila arizonae (Fruit fly) Adh2 P27581 Drosophila buzzatii (Fruit fly) Adh2 P25720 Drosophila hydei (Fruit fly) Adh2 P23237 Drosophila montana (Fruit fly) Adh2 P48587 Drosophila mulleri (Fruit fly) Adh2 P07160 Drosophila wheeleri (Fruit fly) Adh2 P24267 Entamoeba histolytica ADH2 Q24803 Hordeum vulgare (Barley) ADH2 P10847 Kluyveromyces marxianus (Yeast) (Candida kefyr) ADH2 Q9P4C2 Zea mays (Maize) ADH2 P04707 Oryza sativa subsp. indica (Rice) ADH2 Q4R1E8 Solanum lycopersicum (Tomato) (Lycopersicon esculentum) ADH2 P28032 Solanum tuberosum (Potato) ADH2 P14674 Scheffersomyces stipitis (strain ATCC 58785/CBS 6054/ ADH2, ADH1, O13309 NBRC 10063/NRRL Y-11545) (Yeast) (Pichia stipitis) PICST_27980 Arabidopsis thaliana (Mouse-ear cress) ADH2, ADHIII, Q96533 FDH1, At5g43940, MRH10.4 Saccharomyces cerevisiae (strain ATCC 204508/S288c) ADH2, ADR2, P00331 (Baker's yeast) YMR303C, YM9952.05C Candida albicans (strain SC5314/ATCC MYA-2876) ADH2, Ca41C10.04, O94038 (Yeast) CaO19.12579, CaO19.5113 Oryza sativa subsp. japonica (Rice) ADH2, DUPR11.1, Q0ITW7 Os11g0210500, LOC_Os11g10510 Drosophila mojavensis (Fruit fly) Adh2, GI17643 P09369 Kluyveromyces lactis (strain ATCC 8585/CBS 2359/DSM ADH2, P49383 70799/NBRC 1267/NRRL Y-1140/WM37) (Yeast) KLLA0F18260g (Candida sphaerica) Oryctolagus cuniculus (Rabbit) ADH2-1 O46649 Oryctolagus cuniculus (Rabbit) ADH2-2 O46650 Hordeum vulgare (Barley) ADH3 P10848 Solanum tuberosum (Potato) ADH3 P14675 Kluyveromyces lactis (strain ATCC 8585/CBS 2359/DSM ADH3, P49384 70799/NBRC 1267/NRRL Y-1140/WM37) (Yeast) KLLA0B09064g (Candida sphaerica) Saccharomyces cerevisiae (strain ATCC 204508/S288c) ADH3, YMR083W, P07246 (Baker's yeast) YM9582.08 Homo sapiens (Human) ADH4 P08319 Mus musculus (Mouse) Adh4 Q9QYY9 Rattus norvegicus (Rat) Adh4 Q64563 Struthio camelus (Ostrich) ADH4 P80468 Kluyveromyces lactis (strain ATCC 8585/CBS 2359/DSM ADH4, P49385 70799/NBRC 1267/NRRL Y-1140/WM37) (Yeast) KLLA0F13530g (Candida sphaerica) Schizosaccharomyces pombe (strain 972/ATCC 24843) adh4, SPAC5H10.06c Q09669 (Fission yeast) Saccharomyces cerevisiae (strain YJM789) (Baker's yeast) ADH4, ZRG5, A6ZTT5 SCY_1818 Saccharomyces cerevisiae (strain ATCC 204508/S288c) ADH4, ZRG5, P10127 (Baker's yeast) YGL256W, NRC465 Saccharomyces pastorianus (Lager yeast) (Saccharomyces ADH5 Q6XQ67 cerevisiae x Saccharomyces eubayanus) Bos taurus (Bovine) ADH5 Q3ZC42 Equus caballus (Horse) ADH5 P19854 Mus musculus (Mouse) Adh5, Adh-2, Adh2 P28474 Rattus norvegicus (Rat) Adh5, Adh-2, Adh2 P12711 Oryctolagus cuniculus (Rabbit) ADH5, ADH3 O19053 Homo sapiens (Human) ADH5, ADHX, FDH P11766 Dictyostelium discoideum (Slime mold) adh5, Q54TC2 DDB_G0281865 Saccharomyces cerevisiae (strain ATCC 204508/S288c) ADH5, YBR145W, P38113 (Baker's yeast) YBR1122 Homo sapiens (Human) ADH6 P28332 Peromyscus maniculatus (North American deer mouse) ADH6 P41681 Pongo abelii (Sumatran orangutan) (Pongo pygmaeus abelii) ADH6 Q5R7Z8 Rattus norvegicus (Rat) Adh6 Q5XI95 Homo sapiens (Human) ADH7 P40394 Rattus norvegicus (Rat) Adh7 P41682 Mus musculus (Mouse) Adh7, Adh-3, Adh3 Q64437 Mycobacterium tuberculosis (strain CDC 1551/Oshkosh) adhA, MT1911 P9WQC0 Rhizobium meliloti (strain 1021) (Ensifer meliloti) adhA, RA0704, O31186 (Sinorhizobium meliloti) SMa1296 Mycobacterium tuberculosis (strain ATCC 25618/H37Rv) adhA, Rv1862 P9WQC1 Zymomonas mobilis subsp. mobilis (strain ATCC 31821/ adhA ZMO1236 P20368 ZM4/CP4) Mycobacterium bovis (strain ATCC BAA-935/AF2122/97) adhB, Mb0784c Q7U1B9 Mycobacterium tuberculosis (strain CDC 1551/Oshkosh) adhB, MT0786 P9WQC6 Mycobacterium tuberculosis (strain ATCC 25618/H37Rv) adhB, Rv0761c, P9WQC7 MTCY369.06c Zymomonas mobilis subsp. mobilis (strain ATCC 31821/ adhB, ZMO1596 P0DJA2 ZM4/CP4) Zymomonas mobilis subsp. mobilis (strain ATCC 10988/ adhB, Zmob_1541 F8DVL8 DSM 424/LMG 404/NCIMB 8938/NRRL B-806/ZM1) Mycobacterium tuberculosis (strain CDC 1551/Oshkosh) adhD, MT3171 P9WQB8 Mycobacterium tuberculosis (strain ATCC 25618/H37Rv) adhD, Rv3086 P9WQB9 Clostridium acetobutylicum (strain ATCC 824/DSM 792/ adhE, aad, CA_P0162 P33744 JCM 1419/LMG 5710/VKM B-1787) Escherichia coli (strain K12) adhE, ana, b1241, P0A9Q7 JW1228 Escherichia coli O157:H7 adhE, Z2016, P0A9Q8 ECs1741 Rhodobacter sphaeroides (strain ATCC 17023/2.4.1/NCIB adhI, RHOS4_11650, P72324 8253/DSM 158) RSP_2576 Oryza sativa subsp. indica (Rice) ADHIII, OsI_009236 A2XAZ3 Escherichia coli (strain K12) adhP, yddN, b1478, P39451 JW1474 Geobacillus stearothermophilus (Bacillus stearothermophilus) adhT P12311 Emericella nidulans (strain FGSC A4/ATCC 38163/CBS alcA, AN8979 P08843 112.46/NRRL 194/M139) (Aspergillus nidulans) Emericella nidulans (strain FGSC A4/ATCC 38163/CBS alc, AN3741 P54202 112.46/NRRL 194/M139) (Aspergillus nidulans) Emericella nidulans (strain FGSC A4/ATCC 38163/CBS alcC, adh3, AN2286 P07754 112.46/NRRL 194/M139) (Aspergillus nidulans) Arabidopsis thaliana (Mouse-ear cress) At1g22430, F12K8.22 Q9SK86 Arabidopsis thaliana (Mouse-ear cress) At1g22440, F12K8.21 Q9SK87 Arabidopsis thaliana (Mouse-ear cress) At1g32780, F6N18.16 A1L4Y2 Arabidopsis thaliana (Mouse-ear cress) At1g64710, F13O11.3 Q8VZ49 Arabidopsis thaliana (Mouse-ear cress) At4g22110, Q0V7W6 F1N20.210 Arabidopsis thaliana (Mouse-ear cress) At5g24760, Q8LEB2 T4C12_30 Arabidopsis thaliana (Mouse-ear cress) At5g42250, K5J14.5 Q9FH04 Zea mays (Maize) FDH P93629 Drosophila melanogaster (Fruit fly) Fdh, gfd, ODH, P46415 CG6598 Bacillus subtilis (strain 168) gbsB, BSU31050 P71017 Caenorhabditis elegans H24K24.3 Q17335 Oryza sativa subsp. japonica (Rice) Os02g0815500, Q0DWH1 LOC_Os02g57040, OsJ_008550, P0643F09.4 Mycobacterium tuberculosis (strain ATCC 25618/H37Rv) Rv1895 O07737 Caenorhabditis elegans sodh-1, K12G11.3 Q17334 Caenorhabditis elegans sodh-2, K12G11.4 O45687 Pseudomonas sp. terPD P33010 Escherichia coli (strain K12) yiaY, b3589, JW5648 P37686 Moraxella sp. (strain TAE123) P81786 Alligator mississippiensis (American alligator) P80222 Catharanthus roseus (Madagascar periwinkle) (Vinca rosea) P85440 Gadus morhua subsp. callarias (Baltic cod) (Gadus callarias) P26325 Naja naja (Indian cobra) P80512 Pisum sativum (Garden pea) P12886 Pelophylax perezi (Perez's frog) (Rana perezi) P22797 Saara hardwickii (Indian spiny-tailed lizard) (Uromastyx P25405 hardwickii) Saara hardwickii (Indian spiny-tailed lizard) (Uromastyx P25406 hardwickii) Equus caballus (Horse) P00327 Equus caballus (Horse) P00328 Geobacillus stearothermophilus (Bacillus stearothermophilus) P42328 Gadus morhua (Atlantic cod) P81600 Gadus morhua (Atlantic cod) P81601 Myxine glutinosa (Atlantic hagfish) P80360 Octopus vulgaris (Common octopus) P81431 Pisum sativum (Garden pea) P80572 Saara hardwickii (Indian spiny-tailed lizard) (Uromastyx P80467 hardwickii) Scyliorhinus canicula (Small-spotted catshark) (Squalus P86884 canicula) Sparus aurata (Gilthead sea bream) P79896

In some embodiments, an α-dioxygenase is used to catalyze the conversion of a fatty acid to a fatty aldehyde (Hamberg et al. 2005). Alpha-dioxygenases catalyze the conversion of a C_(n) fatty acid to a C_(n-1) aldehyde and may serve as an alternative to both ADH and AOX for fatty aldehyde production if a fatty acid is used as a biotransformation substrate. Due to the chain shortening of the dioxygenase reaction, this route requires a different synthesis pathway compared to the ADH and AOX routes. Biotransformations of E. coli cells expressing a rice α-dioxygenase exhibited conversion of C10, C12, C14 and C16 fatty acids to the corresponding C_(n-1) aldehydes. With the addition of the detergent Triton X 100, 3.7 mM of pentadecanal (0.8 g/L) was obtained after 3 hours from hexadecanoic acid with 74% conversion (Kaehne et al. 2011). Exemplary α-dioxygenases are shown in Table 10.

TABLE 10 Exemplary alpha-dioxygenases Entry Organism Gene names Q9SGH6 Arabidopsis thaliana (Mouse-ear cress) DOX1 DIOX1 PADOX-1 PIOX At3g01420 T13O15.6 Q9C9U3 Arabidopsis thaliana (Mouse-ear cress) DOX2 DIOX2 At1g73680 F25P22.10 P14550 Homo sapiens (Human) AKR1A1 ALDR1 ALR Q69EZ9 Solanum lycopersicum (Tomato) (Lycopersicon LOC543896 esculentum) Q5WM33 Solanum lycopersicum (Tomato) (Lycopersicon alpha-DOX2 esculentum) Q69F00 Solanum lycopersicum (Tomato) (Lycopersicon esculentum) D7LAG3 Arabidopsis lyrata subsp. lyrata (Lyre-leaved ALPHA-DOX1 ARALYDRAFT_317048 rock-cress) D8LJL3 Ectocarpus siliculosus (Brown alga) DOX Esi_0026_0091 E3U9P5 Nicotiana attenuata (Coyote tobacco) adox2

An enzyme's total turnover number (or TTN) refers to the maximum number of molecules of a substrate that the enzyme can convert before becoming inactivated. In general, the TTN for the hydroxylases and other enzymes used in the methods of the invention range from about 1 to about 100,000 or higher. For example, the TTN can be from about 1 to about 1,000, or from about 1,000 to about 10,000, or from about 10,000 to about 100,000, or from about 50,000 to about 100,000, or at least about 100,000. In particular embodiments, the TTN can be from about 100 to about 10,000, or from about 10,000 to about 50,000, or from about 5,000 to about 10,000, or from about 1,000 to about 5,000, or from about 100 to about 1,000, or from about 250 to about 1,000, or from about 100 to about 500, or at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, 100,000, or more.

When whole cells expressing a hydroxylase are used to carry out a hydroxylation reaction, the turnover can be expressed as the amount of substrate that is converted to product by a given amount of cellular material. In general, in vivo hydroxylation reactions exhibit turnovers from at least about 0.01 to at least about 10 mmol·g_(cdw) ⁻¹, wherein g_(cdw) is the mass of cell dry weight in grams. When whole cells expressing a hydroxylase are used to carry out a hydroxylation reaction, the activity can further be expressed as a specific productivity, e.g., concentration of product formed by a given concentration of cellular material per unit time, e.g., in g/L of product per g/L of cellular material per hour (g g_(cdw) ⁻¹ h⁻¹). In general, in vivo hydroxylation reactions exhibit specific productivities from at least about 0.01 to at least about 0.5 g·g_(cdw) ⁻¹ h⁻¹, wherein g_(cdw) is the mass of cell dry weight in grams.

The TTN for heme enzymes, in particular, typically ranges from about 1 to about 100,000 or higher. For example, the TTN can be from about 1 to about 1,000, or from about 1,000 to about 10,000, or from about 10,000 to about 100,000, or from about 50,000 to about 100,000, or at least about 100,000. In particular embodiments, the TTN can be from about 100 to about 10,000, or from about 10,000 to about 50,000, or from about 5,000 to about 10,000, or from about 1,000 to about 5,000, or from about 100 to about 1,000, or from about 250 to about 1,000, or from about 100 to about 500, or at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, 100,000, or more. In certain embodiments, the variant or chimeric heme enzymes of the present invention have higher TTNs compared to the wild-type sequences. In some instances, the variant or chimeric heme enzymes have TTNs greater than about 100 (e.g., at least about 100, 150, 200, 250, 300, 325, 350, 400, 450, 500, or more) in carrying out in vitro hydroxylation reactions. In other instances, the variant or chimeric heme enzymes have TTNs greater than about 1000 (e.g., at least about 1000, 2500, 5000, 10,000, 25,000, 50,000, 75,000, 100,000, or more) in carrying out in vivo whole cell hydroxylation reactions.

When whole cells expressing a heme enzyme are used to carry out a hydroxylation reaction, the turnover can be expressed as the amount of substrate that is converted to product by a given amount of cellular material. In general, in vivo hydroxylation reactions exhibit turnovers from at least about 0.01 to at least about 10 mmol·g_(cdw) ⁻¹, wherein g_(cdw) is the mass of cell dry weight in grams. For example, the turnover can be from about 0.1 to about 10 mmol·g_(cdw) ⁻¹, or from about 1 to about 10 mmol·g_(cdw) ⁻¹, or from about 5 to about 10 mmol·g_(cdw) ⁻¹, or from about 0.01 to about 1 mmol·g_(cdw) ⁻¹, or from about 0.01 to about 0.1 mmol·g_(cdw) ⁻¹, or from about 0.1 to about 1 mmol·g_(cdw) ⁻¹, or greater than 1 mmol·g_(cdw) ⁻¹. The turnover can be about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or about 10 mmol·g_(cdw) ⁻¹.

When whole cells expressing a heme enzyme are used to carry out a hydroxylation reaction, the activity can further be expressed as a specific productivity, e.g., concentration of product formed by a given concentration of cellular material per unit time, e.g., in g/L of product per g/L of cellular material per hour (g·g_(cdw) ⁻¹ h⁻¹). In general, in vivo hydroxylation reactions exhibit specific productivities from at least about 0.01 to at least about 0.5 g·g_(cdw) ⁻¹ h⁻¹, wherein g_(cdw) is the mass of cell dry weight in grams. For example, the specific productivity can be from about 0.01 to about 0.1 g·g_(cdw) ⁻¹ h⁻¹, or from about 0.1 to about 0.5 g·g_(cdw) ¹ h⁻¹, or greater than 0.5 g·g_(cdw) ⁻¹ h⁻¹. The specific productivity can be about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or about 0.5 g·g_(cdw) ⁻¹ h⁻¹.

In certain embodiments, mutations can be introduced into the target gene using standard cloning techniques (e.g., site-directed mutagenesis) or by gene synthesis to produce the hydroxylases (e.g., cytochrome P450 variants) of the present invention. The mutated gene can be expressed in a host cell (e.g., bacterial cell) using an expression vector under the control of an inducible promoter or by means of chromosomal integration under the control of a constitutive promoter. Hydroxylation activity can be screened in vivo or in vitro by following product formation by GC or HPLC as described herein.

The expression vector comprising a nucleic acid sequence that encodes a heme enzyme of the invention can be a viral vector, a plasmid, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage (e.g., a bacteriophage P1-derived vector (PAC)), a baculovirus vector, a yeast plasmid, or an artificial chromosome (e.g., bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a mammalian artificial chromosome (MAC), and human artificial chromosome (HAC)). Expression vectors can include chromosomal, non-chromosomal, and synthetic DNA sequences. Equivalent expression vectors to those described herein are known in the art and will be apparent to the ordinarily skilled artisan.

The expression vector can include a nucleic acid sequence encoding a heme enzyme that is operably linked to a promoter, wherein the promoter comprises a viral, bacterial, archaeal, fungal, insect, or mammalian promoter. In certain embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In other embodiments, the promoter is a tissue-specific promoter or an environmentally regulated or a developmentally regulated promoter.

It is to be understood that affinity tags may be added to the N- and/or C-terminus of a heme enzyme expressed using an expression vector to facilitate protein purification. Non-limiting examples of affinity tags include metal binding tags such as His6-tags and other tags such as glutathione S-transferase (GST).

Non-limiting expression vectors for use in bacterial host cells include pCWori, pET vectors such as pET22 (EMD Millipore), pBR322 (ATCC37017), pQE™ vectors (Qiagen), pBluescript™ vectors (Stratagene), pNH vectors, lambda-ZAP vectors (Stratagene); ptrc99a, pKK223-3, pDR540, pRIT2T (Pharmacia), pRSET, pCR-TOPO vectors, pET vectors, pSyn_1 vectors, pChlamy_1 vectors (Life Technologies, Carlsbad, Calif.), pGEM1 (Promega, Madison, Wis.), and pMAL (New England Biolabs, Ipswich, Mass.). Non-limiting examples of expression vectors for use in eukaryotic host cells include pXT1, pSGS (Stratagene), pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia), pcDNA3.3, pcDNA4/TO, pcDNA6/TR, pLenti6/TR, pMT vectors (Life Technologies), pKLAC1 vectors, pKLAC2 vectors (New England Biolabs), pQE™ vectors (Qiagen), BacPak baculoviral vectors, pAdeno-X™ adenoviral vectors (Clontech), and pBABE retroviral vectors. Any other vector may be used as long as it is replicable and viable in the host cell.

The host cell can be a bacterial cell, an archaeal cell, a fungal cell, a yeast cell, an insect cell, or a mammalian cell.

Suitable bacterial host cells include, but are not limited to, BL21 E. coli, DE3 strain E. coli, E. coli M15, DH5α, DH10β, HB101, T7 Express Competent E. coli (NEB), B. subtilis cells, Pseudomonas fluorescens cells, and cyanobacterial cells such as Chlamydomonas reinhardtii cells and Synechococcus elongates cells. Non-limiting examples of archaeal host cells include Pyrococcus furiosus, Metallosphera sedula, Thermococcus litoralis, Methanobacterium thermoautotrophicum, Methanococcus jannaschii, Pyrococcus abyssi, Sulfolobus solfataricus, Pyrococcus woesei, Sulfolobus shibatae, and variants thereof. Fungal host cells include, but are not limited to, yeast cells from the genera Saccharomyces (e.g., S. cerevisiae), Pichia (P. Pastoris), Kluyveromyces (e.g., K. lactis), Hansenula and Yarrowia, and filamentous fungal cells from the genera Aspergillus, Trichoderma, and Myceliophthora. Suitable insect host cells include, but are not limited to, Sf9 cells from Spodoptera frugiperda, Sf21 cells from Spodoptera frugiperda, Hi-Five cells, BTI-TN-5B1-4 Trichophusia ni cells, and Schneider 2 (S2) cells and Schneider 3 (S3) cells from Drosophila melanogaster. Non-limiting examples of mammalian host cells include HEK293 cells, HeLa cells, CHO cells, COS cells, Jurkat cells, NS0 hybridoma cells, baby hamster kidney (BHK) cells, MDCK cells, NIH-3T3 fibroblast cells, and any other immortalized cell line derived from a mammalian cell.

In certain embodiments, the present invention provides heme enzymes such as the P450 variants described herein that are active hydroxylation catalysts inside living cells. As a non-limiting example, bacterial cells (e.g., E. coli) can be used as whole cell catalysts for the in vivo hydroxylation reactions of the present invention. In some embodiments, whole cell catalysts containing P450 enzymes with the equivalent C400× mutation are found to significantly enhance the total turnover number (TTN) compared to in vitro reactions using isolated P450 enzymes.

Biohydroxylation Reaction Conditions

The methods of the invention include forming reaction mixtures that contain the hydroxylases described herein. The hydroxylases can be, for example, purified prior to addition to a reaction mixture or secreted by a cell present in the reaction mixture. The reaction mixture can contain a cell lysate including the enzyme, as well as other proteins and other cellular materials. Alternatively, a hydroxylase can catalyze the reaction within a cell expressing the hydroxylase. Any suitable amount of hydroxylase can be used in the methods of the invention. In general, hydroxylation reaction mixtures contain from about 0.01 weight % (wt %) to about 100 wt % hydroxylase with respect to the hydrocarbon substrate. The reaction mixtures can contain, for example, from about 0.01 wt % to about 0.1 wt % hydroxylase, or from about 0.1 wt % to about 1 wt % hydroxylase, or from about 1 wt % to about 10 wt % hydroxylase, or from about 10 wt % to about 100 wt % hydroxylase. The reaction mixtures can contain from about 0.05 wt % to about 5 wt % hydroxylase, or from about 0.05 wt % to about 0.5 wt % hydroxylase. The reaction mixtures can contain about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, or about 3 wt % hydroxylase. One of skill in the art will understand how to convert wt % values to mol % values with respect to the hydroxylase and/or substrate concentrations set forth herein.

If the hydroxylase catalyses the reaction within a cell expressing the hydroxylase then any suitable amount of cells can be used in the methods of the invention. In general, hydroxylation whole-cell reaction mixtures contain from about about 1 weight % to about 10,000 wt % of cells on a cell dry weight basis with respect to the hydrocarbon substrate. The whole-cell reaction mixtures can contain, for example, from about 1 wt % to about 10 wt % cells, or from about 10 wt % to about 100 wt % cells, or from about 100 wt % to about 1000 wt % cells, or from about 1000 wt % cells to about 2500 wt % cells, or from about 2500 wt % cells to about 5000 wt % cells, or from about 5000 wt % cells to about 7500 wt % cells, or from about 7500 wt % cells to about 10000 wt % cells with respect to the hydrocarbon substrate. The whole-cell reaction mixtures can contain from about 2 wt % to about 1000 wt % cells, or from about 5 wt % to about 500 wt % cells with respect to the hydrocarbon substrate. The whole-cell reaction mixtures can contain about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or about 1000 wt % cells with respect to the hydrocarbon substrate.

The concentration of a saturated or unsaturated hydrocarbon substrate is typically in the range of from about 100 μM to about 1 M. The concentration can be, for example, from about 100 μM to about 1 mM, or about from 1 mM to about 100 mM, or from about 100 mM to about 500 mM, or from about 500 mM to 1 M. The concentration can be from about 500 μM to about 500 mM, 500 μM to about 50 mM, or from about 1 mM to about 50 mM, or from about 15 mM to about 45 mM, or from about 15 mM to about 30 mM. The concentration of the saturated or unsaturated hydrocarbon substrate can be, for example, about 100, 200, 300, 400, 500, 600, 700, 800, or 900 μM. The concentration of the saturated or unsaturated hydrocarbon substrate can be about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mM.

Reaction mixtures can contain additional reagents. As non-limiting examples, the reaction mixtures can contain buffers (e.g., 2-(N-morpholino)ethanesulfonic acid (MES), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), 3-morpholinopropane-1-sulfonic acid (MOPS), 2-amino-2-hydroxymethyl-propane-1,3-diol (TRIS), potassium phosphate, sodium phosphate, phosphate-buffered saline, sodium citrate, sodium acetate, and sodium borate), cosolvents (e.g., dimethylsulfoxide, dimethylformamide, ethanol, methanol, isopropanol, glycerol, tetrahydrofuran, acetone, acetonitrile, and acetic acid), salts (e.g., NaCl, KCl, CaCl₂, and salts of Mn²⁺ and Mg²⁺), denaturants (e.g., urea and guandinium hydrochloride), detergents (e.g., sodium dodecylsulfate and Triton-X 100), chelators (e.g., ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), 2-({2-[Bis(carboxymethyl)amino]ethyl}(carboxymethyl)amino)acetic acid (EDTA), and 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA)), sugars (e.g., glucose, sucrose, and the like), and reducing agents (e.g., sodium dithionite, NADPH, dithiothreitol (DTT), β-mercaptoethanol (BME), and tris(2-carboxyethyl)phosphine (TCEP)). Buffers, cosolvents, salts, denaturants, detergents, chelators, sugars, and reducing agents can be used at any suitable concentration, which can be readily determined by one of skill in the art. In general, buffers, cosolvents, salts, denaturants, detergents, chelators, sugars, and reducing agents, if present, are included in reaction mixtures at concentrations ranging from about 1 μM to about 1 M. For example, a buffer, a cosolvent, a salt, a denaturant, a detergent, a chelator, a sugar, or a reducing agent can be included in a reaction mixture at a concentration of about 1 μM, or about 10 μM, or about 100 μM, or about 1 mM, or about 10 mM, or about 25 mM, or about 50 mM, or about 100 mM, or about 250 mM, or about 500 mM, or about 1 M. Cosolvents, in particular, can be included in the reaction mixtures in amounts ranging from about 1% v/v to about 75% v/v, or higher. A cosolvent can be included in the reaction mixture, for example, in an amount of about 5, 10, 20, 30, 40, or 50% (v/v).

Reactions are conducted under conditions sufficient to catalyze the formation of a hydroxylation product. The reactions can be conducted at any suitable temperature. In general, the reactions are conducted at a temperature of from about 4° C. to about 40° C. The reactions can be conducted, for example, at about 25° C. or about 37° C. The reactions can be conducted at any suitable pH. In general, the reactions are conducted at a pH of from about 3 to about 10. The reactions can be conducted, for example, at a pH of from about 6.5 to about 9. The reactions can be conducted for any suitable length of time. In general, the reaction mixtures are incubated under suitable conditions for anywhere between about 1 minute and several hours. The reactions can be conducted, for example, for about 1 minute, or about 5 minutes, or about 10 minutes, or about 30 minutes, or about 1 hour, or about 2 hours, or about 4 hours, or about 8 hours, or about 12 hours, or about 24 hours, or about 48 hours, or about 72 hours, or about 96 hours, or about 120 hours, or about 144 hours, or about 168 hours, or about 192 hours. In general, reactions are conducted under aerobic conditions. In some embodiments, the solvent forms a second phase, and the hydroxylation occurs in the aqueous phase. In some embodiments, the hydroxylases is located in the aqueous layer whereas the substrates and/or products occur in an organic layer. Other reaction conditions may be employed in the methods of the invention, depending on the identity of a particular hydroxylase, or olefinic substrate.

Reactions can be conducted in vivo with intact cells expressing a hydroxylase of the invention. The in vivo reactions can be conducted with any of the host cells used for expression of the hydroxylases, as described herein. A suspension of cells can be formed in a suitable medium supplemented with nutrients (such as mineral micronutrients, glucose and other fuel sources, and the like). Hydroxylation yields from reactions in vivo can be controlled, in part, by controlling the cell density in the reaction mixtures. Cellular suspensions exhibiting optical densities ranging from about 0.1 to about 50 at 600 nm can be used for hydroxylation reactions. Other densities can be useful, depending on the cell type, specific hydroxylases, or other factors.

Pheromone Compositions and Uses Thereof

As described above, many of the olefinic alcohol products made via the methods described herein are pheromones. Pheromones prepared according to the methods of the invention can be formulated for use as insect control compositions. The pheromone compositions can include a carrier, and/or be contained in a dispenser. The carrier can be, but is not limited to, an inert liquid or solid.

Examples of solid carriers include but are not limited to fillers such as kaolin, bentonite, dolomite, calcium carbonate, talc, powdered magnesia, Fuller's earth, wax, gypsum, diatomaceous earth, rubber, plastic, silica and China clay. Examples of liquid carriers include, but are not limited to, water; alcohols, such as ethanol, butanol or glycol, as well as their ethers or esters, such as methylglycol acetate; ketones, such as acetone, cyclohexanone, methylethyl ketone, methylisobutylketone, or isophorone; alkanes such as hexane, pentane, or heptanes; aromatic hydrocarbons, such as xylenes or alkyl naphthalenes; mineral or vegetable oils; aliphatic chlorinated hydrocarbons, such as trichloroethane or methylene chloride; aromatic chlorinated hydrocarbons, such as chlorobenzenes; water-soluble or strongly polar solvents such as dimethylformamide, dimethyl sulfoxide, or N-methylpyrrolidone; liquefied gases; and mixtures thereof. Baits or feeding stimulants can also be added to the carrier.

Pheromone compositions can be formulated so as to provide slow release into the atmosphere, and/or so as to be protected from degradation following release. For example, the pheromone compositions can be included in carriers such as microcapsules, biodegradable flakes and paraffin wax-based matrices.

Pheromone compositions can contain other pheromones or attractants provided that the other compounds do not substantially interfere with the activity of the composition. The pheromone compositions can also include insecticides. Examples of suitable insecticides include, but are not limited to, buprofezin, pyriproxyfen, flonicamid, acetamiprid, dinotefuran, clothianidin, acephate, malathion, quinolphos, chloropyriphos, profenophos, bendiocarb, bifenthrin, chlorpyrifos, cyfluthrin, diazinon, pyrethrum, fenpropathrin, kinoprene, insecticidal soap or oil, and mixtures thereof.

Pheromone compositions can be used in conjuction with a dispenser for release of the composition in a particular environment. Any suitable dispenser known in the art can be used. Examples of such dispensers include but are not limited to bubble caps comprising a reservoir with a permeable barrier through which pheromones are slowly released, pads, beads, tubes rods, spirals or balls composed of rubber, plastic, leather, cotton, cotton wool, wood or wood products that are impregnated with the pheromone composition. For example, polyvinyl chloride laminates, pellets, granules, ropes or spirals from which the pheromone composition evaporates, or rubber septa. One of skill in the art will be able to select suitable carriers and/or dispensers for the desired mode of application, storage, transport or handling.

A variety of pheromones, including those set forth in Table 1 can be prepared according to the methods of the invention and formulated as described above. For example, the methods of the invention can be used to prepare peach twig borer (PTB) sex pheromone, which is a mixture of (E)-dec-5-en-1-ol (17%) and (E)-dec-5-en-1-yl acetate (83%). The PTB sex pheromone can be used in conjunction with a sustained pheromone release device having a polymer container containing a mixture of the PTB sex pheromone and a fatty acid ester (such as a sebacate, laurate, palmitate, stearate or arachidate ester) or a fatty alcohol (such as undecanol, dodecanol, tridecanol, tridecenol, tetradecanol, tetradecenol, tetradecadienol, pentadecanol, pentadecenol, hexadecanol, hexadecenol, hexadecadienol, octadecenol and octadecadienol). The polymer container can be a tube, an ampul, or a bag made of a polyolefin or an olefin component-containing copolymer. Sex pheromones of other pest insects such the cotton bollworm (Helicoverpa armigera), fall army worm (Spodoptera frugiperda), oriental fruit moth (Grapholita molesta) and leaf roller (Tortricidae) can be used in this type of sustained pheromone release device. The sex pheromones typically include one or more aliphatic acetate compounds having from 10 to 16 carbon atoms (e.g., decyl acetate, decenyl acetate, decadienyl acetate, undecyl acetate, undecenyl acetate, dodecyl acetate, dodecenyl acetate, dodecadienyl acetate, tridecyl acetate, tridecenyl acetate, tridecadienyl acetate, tetradecyl acetate, tetradecenyl acetate, tetradecadienyl acetate, and the like) and/or one or more aliphatic aldehyde compounds having from 10 to 16 carbon atoms (e.g., 7-hexadecenal, 11-hexadecenal, 13-octadecenal, and the like).

Pheromones prepared according to the methods of the invention, as well as compositions containing the pheromones, can be used to control the behavior and/or growth of insects in various environments. The pheromones can be used, for example, to attract or repel male or female insects to or from a particular target area. The pheromones can be used to attract insects away from vulnerable crop areas. The pheromones can also be used example to attract insects as part of a strategy for insect monitoring, mass trapping, lure/attract-and-kill or mating disruption.

Mass trapping involves placing a high density of traps in a crop to be protected so that a high proportion of the insects are removed before the crop is damaged. Lure/attract-and-kill techniques are similar except once the insect is attracted to a lure, it is subjected to a killing agent. Where the killing agent is an insecticide, a dispenser can also contain a bait or feeding stimulant that will entice the insects to ingest an effective amount of the insecticide.

It will be appreciated by a person skilled in the art that a variety of different traps are possible. Suitable examples of such traps include water traps, sticky traps, and one-way traps. Sticky traps come in many varieties. One example of a sticky trap is of cardboard construction, triangular or wedge-shaped in cross-section, where the interior surfaces are coated with a non-drying sticky substance. The insects contact the sticky surface and are caught. Water traps include pans of water and detergent that are used to trap insects. The detergent destroys the surface tension of the water, causing insects that are attracted to the pan, to drown in the water. One-way traps allow an insect to enter the trap but prevent it from exiting. The traps of the invention can be colored brightly, to provide additional attraction for the insects.

The trap is positioned in an area infested (or potentially infested) with insects. Generally, the trap is placed on or close to a tree or large plant. The aroma of the pheromone attracts the insects to the trap. The insects can then be caught, immobilised and/or killed within the trap, for example, by the killing agent present in the trap.

Pheromones prepared according to the methods of the invention can also be used to disrupt mating. Strategies of mating disruption include confusion, trail-masking and false-trail following. Constant exposure of insects to a high concentration of a pheromone can prevent male insects from responding to normal levels of the pheromone released by female insects. Trail-masking uses a pheromone to destroy the trail of pheromones released by females. False-trail following is carried out by laying numerous spots of a pheromone in high concentration to present the male with many false trails to follow. When released in sufficiently high quantities, the male insects are unable to find the natural source of the sex pheromones (the female insects) so that mating cannot occur.

Insect populations can be surveyed or monitored by counting the number of insects in a target area (e.g., the number of insects caught in a trap). Inspection by a horticulturist can provide information about the life stage of a population. Knowing where insects are, how many of them there are, and their life stage enables informed decisions to be made as to where and when insecticides or other treatments are warranted. For example, a discovery of a high insect population can necessitate the use of methods for removal of the insect. Early warning of an infestation in a new habitat can allow action to be taken before the population becomes unmanageable. Conversely, a discovery of a low insect population can lead to a decision that it is sufficient to continue monitoring the population. Insect populations can be monitored regularly so that the insects are only controlled when they reach a certain threshold. This provides cost-effective control of the insects and reduces the environmental impact of the use of insecticides.

As will be apparent to one of skill in the art, the amount of a pheromone or pheromone composition used for a particular application can vary depending on several factors such as the type and level of infestation; the type of composition used; the concentration of the active components; how the composition is provided, for example, the type of dispenser used; the type of location to be treated; the length of time the method is to be used for; and environmental factors such as temperature, wind speed and direction, rainfall and humidity. Those of skill in the art will be able to determine an effective amount of a pheromone or pheromone composition for use in a given application.

EXAMPLES

The present invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of noncritical parameters which can be changed or modified to yield essentially the same results.

General Methods.

E. coli heat shock transformation: Heat shock transformation of plasmids/Gibson assembly products/ligation products were performed with competent BL21 (DE3) cells from NEB (C2527H). Set a water bath to 42° C. and remove the SOC media from storage at 4′C and incubate at 37′C. Remove chemically competent cells from the −80° C. freezer and thaw on ice for 5-10 minutes. Add the DNA solution to be transformed to the cells and tap the tube gently to mix, keep the mixture on ice for 5 more minutes. Submerge the tube containing the cell/DNA mixture in the 42° C. water bath for 10 seconds and quickly place the tubes back on ice for 2 minutes. After the two minute incubation on ice, add 500 μl of SOC recovery media and incubate at 37° C. with 210 rpm shaking for 60 minutes. After incubation, 50-200 μl of the recovered cells are plated on LB/agar plates supplemented with the appropriate antibiotics.

E. coli plasmid isolation: Plasmid isolation of 2.5 ml overnight culture were performed with the SpinSmart™ Plasmid Miniprep DNA Purification kit (Danville Scientific). The overnight culture is collected by centrifugation at 14,000 rpm for 10 minutes. Resuspend the pellet in 250 μl of P1 buffer and transfer the mixture to a 1.5 ml microcentrifuge tube. Add 250 μl of P2 buffer and mix by inverting the tube. Add 300 μl of P3 buffer and mix by inverting the tube. Centrifuge the mixture at 14,000 rpm for 5 minutes and transfer the supernatant containing DNA onto a fresh plasmid binding spin column. Bind DNA onto the column by centrifugation at 14,000 rpm for 30 seconds, followed by washes with 500 μl of P4 wash buffer and 650 μl of P5 wash buffer. Dry the column by centrifugation at 14,000 rpm for 2 minutes and then discard the collection tube. Place the plasmid binding spind column in a 1.5 ml microcentrifuge tube. Add 50 μl of warm DI water (50° C.) and centrifuge at 14000 rpm to elute the DNA.

E. coli DNA isolation: DNA fragment isolation from agarose gel was performed with the Zymoclean Gel DNA Recovery Kits (Zymo research). Excise the desired gel fragment using a razor blade and transfer it to a 1.5 ml microcentrifuge tube. Add 250 μl of ADB to the tube and incubate at 50° C. for 5 to 10 minutes until the gel slice is dissolved. Transfer the melted agarose solution to a Zymo-spin™ column in a collection tube, centrifuge for 30-60 seconds and discard the flow through. Wash the column with 200 μl of DNA wash buffer and centrifuge for 30 seconds. Repeat the wash step. Dry the column by centrifugation at 14,000 rpm for 2 minutes. Place the Zymo-spin™ column in a 1.5 ml microcentrifuge tube. Add 12 μl of warm DI water (50° C.) and centrifuge at 14000 rpm to elute DNA.

PCR: PCR reactions were performed using Phusion High-Fidelity PCR Master Mix with HF Buffer (NEB: M0531S). In a typical 25 μl PCR reaction, 0.5 μl of the template DNA (plasmid, PCR product, synthesized DNA, etc) is mixed with 1.25 μl of each of the forward and reverse primers (10 mM) along with 0.75 μl of DMSO, 12.5 μl of the Phusion master mix and 8.75 μl of DI water. A typical thermocycler program consists of the follow steps:

Temperature Time Step (° C.) (s) 1 98 30 2 98 10 Repeat 3 55 10 25X 4 72 50 5 72 600 6 10 ∞

SOE-PCR: A typical 50 μl reaction contains (1) 0.5 to 3 μl of each isolated DNA fragment being spliced (2) 2.5 μl of each the forward and reverse primers (10 mM), (3) 1.5 μl of DMSO, (4) 25 μl of Phusion master mix and (5) enough DI water to reach a final total volume of 50 μl. The thermocycler program is modified from that of table # by extending the time of step 3 to 20 seconds.

E. coli biotransformations: Each E. coli strain expressing a terminal hydroxylase as well as the necessary redox partners was cultured in shake flasks to produce cell mass and induce protein expression. At the end of the protein expression phase, the cells were collected by centrifugation and washed with the bioconversion buffer. The washed cells were resuspended in bioconversion buffer to yield a cell density of ˜100 g cell wet weight (g cww)/L. A volume of 1 ml of this cell mixture was transferred to a 20 ml amber vial. The bioconversion was initiated by the addition of 200 μl of a 50/50 (v/v) mixture of substrate and isopropyl alcohol. For E/Z-5-decene and 1-bromodecane, the reactions were quenched after 1 hour with the addition of 100 μl 3 M HCl and analyzed for product formation with GC/FID. For 1-dodecene, 1-dodecyne, and hexadecane, the reactions were quenched after 4 hours with HCl and analyzed with GC/FID.

Example 1 Synthesis of Peach Twig Borer (PTB) Sex Pheromone Using Biocatalytic Terminal Hydroxylation

The PTB pheromone is a mixture of (E)-dec-5-en-1-ol (17%) and (E)-dec-5-en-1-yl acetate (83%). Using a terminal hydroxylase described in Table 3, 4, or 5, (E)-dec-5-ene can be readily converted into (E)-dec-5-en-1-ol, which can be acetylated to yield (E)-dec-5-en-1-yl acetate, as shown in Scheme 22. A suitable terminal hydroxylase enables this original synthesis route and reduces the synthesis cost of this pheromone.

Example 2 Biocatalytic Hydroxylation of Asymmetric and Diolefin Substrates

Asymmetric substrates shown in Tables 11, 12, and 13, including (Z)-hexadec-11-ene and diolefin substrates such as (E,E)-8,10-dodecadiene, are terminally hydroxylated via the enzymes presented in Tables 3, 4, and 5.

TABLE 11 Production of representative alkenols enabled by a terminal hydroxylase with activity for alkenes with internal olefins. Biological importance Substrate (alkene) Product (alkenol) of Product

  (Z)-non-3-ene

  (Z)-non-3-en-1-ol West indian fruit fly sex pheromone

  (E)-dec-5-ene

  (E)-dec-5-en-1-ol Peach twig borer sex pheromone

  (Z)-dodec-8-ene

  (Z)-dodec-8-en-12-ol Oriental fruit moth sex pheromone

  (E,E)-8,10-Dodecadiene

  (E,E)-8,10-Dodecadien-1-ol Codling moth sex pheromone

TABLE 12 Production of representative alkenals and allylic acetates enabled by a terminal hydroxylase with activity for alkenes with internal olefins. Biological Final product (alkenal or allylic importance Substrate (alkene) acetate) of Product

  (Z)-hexadec-11-ene

  (Z)-hexadec-11-en-1-yl acetate Pandemis leafroller sex pheromone

  (Z)-tetradec-9-ene

  (Z)-hexadec-11-enal

  (Z)-tetradec-9-en-1-yl acetate

  (Z)-hexadec-11-ene

  (Z)-hexadec-9-enal Cotton bollworm sex pheromone

  (Z)-hexadec-9-ene

  (Z)-hexadec-11-enal

  (Z)-tetradec-11-ene

  (Z)-tetradec-11-en-1-yl acetate Oblique banded leafroller sex pheromone

TABLE 13 Production of representative alkenols enabled by a terminal hydroxylase with activity for alkenes with internal olefins. The products listed here are either pheromones in their own right, or are precursors to pheromones, e.g., via oxidation or acetylation. Biological Substrate (alkene) Product (alkenol) importance of Product

  (Z)-3-nonene

  (Z)-3-nonenol West Indian Fruity Fly male sex pheromone

  Z-5-decene

  Z-5-decen-1-ol

  (E)-8-dodecene

  (E)-8-dodecenol Citrus Fruit Moth sex pheromone

  (Z)-8-dodecene

  (Z)-8-dodecenol Oriental Fruit moth sex pheromone component

  Z-7-dodecene

  Z-7-dodecen-1-ol

  Z-9-tetradecene

  Z-9-tetradecen-1-ol

  Z-11-tetradecene

  Z-11-tetradecen-1-ol

  E-11-tetradecene

  E-11-tetradecen-1-ol

  (Z,E)-9,11,13-Tetradecatriene

  (Z,E)-9,11,13-Tetradecatrienol

  Z-7-hexadecene

  Z-7-hexadecen-1-ol

  Z-9-hexadecene

  Z-9-hexadecenol

  Z-11-hexadecene

  Z-11-hexadecenol

  (Z,E)-9,11-Hexadecadiene

  (Z,E)-9,11-Hexadecadien-1-ol

  (Z,Z)-11,13-Hexadecadiene

  (Z,Z)-11,13-Hexadecadien-1-o1 Naval Orangeworm sex pheromone component

  (Z,E)-11,13-Hexadecadiene

  (Z,E)-11,13-Hexadecadien-1-ol Naval Orangeworm sex pheromone component

  Z-13-octadecene

  Z-13-octadecen-1-ol

Example 3 Biocatalyst Identity and/or Construction

The purpose of this example is to illustrate the construction and/or identity of the various biocatalysts described in the present disclosure.

Strains, plasmids, and oligonucleotides disclosed herein are listed in Tables 14-16.

TABLE 14 Genotypes of strains used in the examples. Strain No. Strain host Strain type Plasmid No. SPV001 E. coli BL21(DE3) pPV001 SPV027 E. coli BL21(DE3) pPV024 SPV014 E. coli BL21(DE3) pPV009 SPV015 E. coli BL21(DE3) pPV010 SPV016 E. coli BL21(DE3) pPV011 SPV017 E. coli BL21(DE3) pPV012 SPV028 E. coli BL21(DE3) pPV025 SPV018 E. coli BL21(DE3) pPV013 SPV029 E. coli BL21(DE3) pPV026 SPV019 E. coli BL21(DE3) pPV014 SPV035 E. coli BL21(DE3) pPV028 SPV036 E. coli BL21(DE3) pPV029 SPV020 E. coli BL21(DE3) pPV015 SPV0021 E. coli BL21(DE3) pPV016 SPV0022 E. coli BL21(DE3) pPV017 SPV0025 E. coli BL21(DE3) pPV003 SPV0013 E. coli BL21(DE3) pPV008 SPV0026 E. coli BL21(DE3) pPV004 SPV0012 E. coli BL21(DE3) pPV007 SPV0023 E. coli BL21(DE3) pPV018 SPV0030 E. coli BL21(DE3) pPV020 SPV0031 E. coli BL21(DE3) pPV021 SPV032 E. coli BL21(DE3) pPV022 SPV037 E. coli BL21(DE3) pPV030 SPV033 E. coli BL21(DE3) pPV023

TABLE 15 Plasmids used in the examples. Other inserts Hydroxylase (reductase Plasmid Hydroxylase Accession No. components and Flanking No. Vector insert (protein ID) transporter) restriction sites pPV001 pET28a(+) none n/a none n/a pPV002 pCom10 none n/a none n/a pPV024 pET28a(+) LadA ABO68832 none BamHI, XhoI pPV009 pET28a(+) LadA_GS EU151491 none BamHI, XhoI pPV010 pET28a(+) CYP153A7 AJ850057 A6FDR, A6FDX XbaI, HindIII pPV011 pET28a(+) CYP153 M. sp. AFO66437 aphH, aphI XbaI, HindIII pPV012 pET28a(+) CYP153A13N2 AB206793 RhFred fused XbaI, SacI pPV025 pET28a(+) CYP153A13N3 AB206799 RhFred fused XbaI, SacI pPV013 pET28a(+) CYP153A13N5 AB206801 RhFred fused XbaI, SacI pPV026 pET28a(+) CYP153A13P2 AB206795 RhFred fused XbaI, SacI pPV014 pColaDuet-1 CYP153RE BAE46203 CPR(BM3) fused, NcoI, SacI AlkL pPV028 pColaDuet-1 CYP153CspK31 ABZ69158 CPR(BM3) fused, NcoI, SacI AlkL pPV029 pColaDuet-1 CYP153Plav ABS63384 CPR(BM3) fused, NcoI, SacI AlkL pPV015 pColaDuet-1 CYP153A7 AJ850057 CPR(BM3) fused, NcoI, SacI AlkL pPV016 pColaDuet-1 CYP153A11 AJ850059 CPR(BM3) fused, NcoI, SacI AlkL pPV017 pColaDuet-1 CYP153D2 AJ850060 CPR(BM3) fused, NcoI, SacI AlkL pPV003 pColaDuet-1 CYP153A M. ABM17701 CPR(BM3) fused, n/a aq. (G307A) AlkL pPV008 pET28a(+) CYP153A M. ABM17701 RhFred fused n/a aq. pPV004 pET28a(+) CYP153A M. ABM17701 CPR(BM3) fused n/a aq. (G307A) pPV007 pET28a(+) CYP153A P.Sp ABE47160 CPR(BM3) fused n/a pPV018 pCom10 AlkB YP_009076004 AlkF, AlkG EcoRI/SalI pPV019 pCom10 none n/a AlkT XhoI/XbaI pPV020 pCom10 AlkB YP_009076004 AlkF, AlkG, AlkT n/a pPV021 pCom10 AlkB YP_009076004 AlkF, AlkG, AlkT, n/a AlkL pPV022 pCom10 AlkB P1 CAB51047 AlkF, AlkG, AlkT, n/a AlkL pPV030 pCom10 AlkB1 AB CAC38027 AlkF, AlkG, AlkT, n/a AlkL pPV023 pCom10 AlkB2 AB CAB51047 AlkF, AlkG, AlkT, n/a AlkL

TABLE 16 Oligonucleotide sequences used in the examples. Oligo- SEQ nucleotide Sequence Description ID NO. 1406_provivi AGCCATCA AlkB 1 023 TCATCATC construct ATCACAGC for DNA AGC synthesis 1406_provivi CACTATAG AlkB P1 2 024 GGGAATTG construct TGAGCGGA for DNA TAACAATT synthesis CC 1406_provivi CTATAGGG AlkB1A.p. 3 025 GAATTGTG construct AGCGGATA for DNA ACAATTCC synthesis C 1406_provivi TGGTGGTG AlkB2A.p. 4 026 CTCGAGTG construct CGGCCGCA for DNA AGCTTCTA synthesis ATG oPV001 CTTAAATC Fwd primer 5 TCGTAGCG for alkB ACTAATTT constructs AATAAAAA TTG oPV002 AAACAGAA Rev primer 6 GCTTGGCT for alkB GCAGGTCG constructs oPV003 CTTATTCC fwd primer 7 TGAGGATT from alkG GGTGCTGC C oPV004 GGCAGCAC rev primer 8 CAATCCTC from alkG AGGAATAA G oPV005 GATGCCGC fwd primer 9 TGGATCTG from SfiI GCCTAGA

All strains listed in Table 14 were obtained by transforming their corresponding plasmid into E. coli BL21(DE3) cells using standard heat shock protocol. Briefly, aliquots of competent E. coli BL21(DE3) were thawed on ice and mixed with 1 ng of isolated DNA plasmid. The mixture was immersed in a 42° C. water bath for 10 seconds, followed by incubation at 4° C. for 2 minutes and 37° C. for 30 minutes before plating on Luria-Bertani agar plates with the appropriate antibiotics.

Plasmid pPV001 was obtained directly from EMD Millipore. Plasmids pPV003, pPV004, pPV007, and pPV008 were directly obtained from the Institut fur Technische Biochemie (Stuttgart, Germany). Plasmid pPV002 was obtained from the California Institute of Technology (Pasadena, USA). Plasmids pPV001, pPV003 and pPV002 served as the vector backbone for pET28a(+), pColaDuet-1, and pCom10 based plasmids listed in Table 15. Gene inserts of pPV009, pPV010, pPV011, pPV012, pPV013, pPV014, pPV015, pPV016, pPV017, pPV018, pPV019, pPV020, pPV021, pPV022, pPV023, pPV024, pPV025, pPV026, pPV0028, pPV0029, and pPV030 designed for the expression of terminal hydroxylases and their redox partners were synthesized (Gen9, Inc. Cambridge, Mass.). The inserts of pPV009, pPV010, pPV011, pPV012, pPV013, pPV0014, pPV0015, pPV0016, pPV0017, pPV018, pPV019, pPV024, pPV025, pPV026, pPV0028 and pPV0029 were cloned into the vector by ligation of vector and insert DNA fragments generated by restriction digest at flanking sites indicated in Table 15. Plasmid pPV0020 was produced by ligation of DNA fragments obtained by restriction digest of pPV0018 (9195 bp fragment) and pPV0019 (2399 bp fragment) with XhoI and XbaI. Plasmid pPV021 was produced by Gibson assembly of DNA fragments obtained by (1) restriction digest of pPV0019 (9089 bp fragment) with EcoRI and SalI and (2) PCR product of 1406_provivi 023 with primers oPV001 and oPV002. The pPV022, pPV023 and pPV030 plasmids were produced by Gibson assembly of DNA fragments obtained by (1) restriction digest of pPV0019 (8550 bp fragment) with EcoRI and SfiI and (2) a splicing by overhang extension (SOE)-PCR product. For pPV022, the SOE-PCR was performed using primers oPV001 and oPV005 along with the PCR products from reactions containing (1) 1406_provivi024 with primers oPV001 and oPV004 and (2) pPV021 with primers oPV003 and oPV005. For pPV023, the SOE-PCR was similarly performed using primers oPV001 and oPV005 along with the PCR products from reactions containing (1) 1406_provivi026 with primers oPV001 and oPV004 and (2) pPV021 with primers oPV003 and oPV005. For pPV030, the SOE-PCR was similarly performed using primers oPV001 and oPV005 along with the PCR products from reactions containing (1) 1406_provivi025 with primers oPV001 and oPV004 and (2) pPV0021 with primers oPV003 and oPV005.

Example 4 Identification of Candidate Enzymes/Strains with Known Terminal Alkane Hydroxylation Activity

Enzymes with terminal hydroxylation activity reported in the literature have generally been characterized for their substrate specificity with linear alkanes and/or fatty acids of various lengths. In certain aspects, the present invention relies upon the observation that the presence of the alkene bonds does not affect the regioselectivity and substrate specificity of the hydroxylases. A subset of known terminal hydroxylases selected from Tables 3, 4, and 5 with preference for C₈-C₁₈ substrates is determined. These sequences are evaluated for terminal hydroxylation according to Scheme 23 with substrates shown in Tables 11, 12, and 13, including (E)-dec-5-ene, (Z)-hexadec-11-ene, and (E,E)-8,10-dodecadiene:

Strains containing these types of enzymes that have been previously used for whole-cell terminal hydroxylation of alkanes or fatty acids are shown in Table 6.

To evaluate the activity of candidate enzymes not already present in an expression strain, the appropriate DNA expression vectors are constructed and transformed in the desired hosts. The identified genes are synthesized as gene products or multiple DNA fragments and inserted into vectors that have been previously used for expression, e.g., pET28a(+) for CYP153 and pGEc47 for AlkB, using standard molecular biology techniques. The correct expression vectors are confirmed by DNA sequencing and then introduced into the desired host via transformation to obtain the desired expression strains.

The constructed expression strains are used to determine the in vivo terminal hydroxylation activity of each candidate enzyme for substrates shown in Tables 11, 12, and 13, including (E)-dec-5-ene, (Z)-hexadec-11-ene, and (E,E)-8,10-dodecadiene. Typically, each strain is cultured in a suitable medium, such as Terrific Broth, until a sufficient cell density to induce enzyme expression with Isopropyl β-D-1-thiogalactopyranoside or dicyclopropylketone. After a predetermined expression period, the cells are pelleted by centrifugation to remove the growth media. The biotransformation reaction is performed in nitrogen-free medium such as M9 or phosphate buffer to ensure the cells are in a resting state to maximize the supply of redox cofactors for the hydroxylation reaction. The alkene is present in the reaction as a neat organic overlay or part of the organic phase in a two-phase reaction. To evaluate strains with sufficient replicates, the initial reaction is performed on a 5-25 mL scale in a vial or flask. A carbon source such as glucose or glycerol can also be added to the reaction to support the regeneration of redox cofactors by the cells. The addition of a co-solvent such as bis(2-ethylhexyl) phthalate to improve the extraction of the product to the organic phase can be explored. In addition, reaction parameters such as reaction time, temperature, volume, ratio of organic and aqueous phases, aeration and cell density can be optimized.

The analysis of the whole-cell hydroxylation of substrates shown in Tables 11, 12, and 13, including (E)-dec-5-ene, (Z)-hexadec-11-ene, and (E,E)-8,10-dodecadiene reactions, requires the development of appropriate gas chromatography (GC) methods. The GC analysis of alkane and fatty acid terminal hydroxylation reactions has been performed with both mass spectrometry and flame ionization detectors. Authentic standards for (E)-dec-5-ene, (E)-dec-5-en-1-ol, and potential side-products, (E)-dec-5-ene-1,10-diol, (E)-dec-5-en-1-al, (E)-dec-5-enoic acid and 2,3-dibutyloxirane are obtained for method development and product quantification. Likewise, (Z)-hexadec-11-ene and (E,E)-8,10-dodecadiene and an assortment of likely hydroxylation or epoxidation products of these two molecules are used for method development and product quantification. A procedure for terminating the hydroxylation reaction and extracting the organic phase into a suitable GC solvent is used.

Demonstration of gram-scale production of (E)-dec-5-en-1-ol using biocatalytic terminal hydroxylation is performed in biotransformation reactions on a liter scale. To efficiently perform whole-cell hydroxylation on a liter scale, the reaction pH and aeration are controlled.

Evaluation of the most promising candidates from the 5-25 mL screening reactions in fermenters enables the identification and alleviation of reaction bottle-necks and inhibition effects of the product and by-products. Enzyme expression can also be further optimized in fermenters with enhanced control over culturing conditions.

Example 5 Hydroxylation of (E)-5-decene and (Z)-5-decene

The purpose of this example is to illustrate the biocatalytic hydroxylation of (E)-5-decene and (Z)-5-decene to (E)-5-decen-1-ol and (Z)-5-decen-1-ol, respectively, and the selectivity of the various enzymes towards either (E)-5-decene or (Z)-5-decene.

The 25 strains detailed in Example 3 were characterized for their ability to convert a mixture of (E)-5-decene and (Z)-5-decene to (E)-5-decen-1-ol and (Z)-5-decen-1-ol in whole cell bioconversion reactions.

Overnight cultures of these 25 strains were inoculated from single colonies grown on LB agar plates containing 30 μg/ml of kanamycin into 2.5 mL of LB medium containing 30 μg/ml of kanamycin and incubated for 24 hours at 37° C. and 210 rpm. The overnight culture was used to inoculate 50 or 100 ml of Terrific Broth with a starting OD₆₀₀ of 0.1. After incubation for approximately 2.5 hours at 37° C. and 210 rpm the cultures reached an OD600 of approximately 1.0-1.5, at which point they were induced with 0.5 mM IPTG and supplemented with 0.5 mM 5-aminolevulinic acid, 50 mg/L thiamine, 1.2 mM MgSO₄, and 25 mL of a solution of trace elements (190 mg CaCl₂*2H₂O, 90 mg ZnSO₄*7H₂O, 90 mg CoCl₂*6H₂O, 75 mg CuSO₄*5H₂O, 50 mg MnSO₄*H₂O, 11.1 mg Na_(z)-EDTA*2H₂O and 8.35 mg FeCl₃*6H₂O in 500 ml of ddH₂O). The culture was further incubated for 20 hours at 20° C. and 180 rpm. The cultures were then pelleted via centrifugation at 3900×g at 4° C. for 10 min, washed once with bioconversion buffer (100 mM phosphate buffer (pH7.2), 1% glycerol/0.4% glucose, 100 μg/ml FeSO4*7H2O, and 30 μg/ml kanamycin), and pelleted again. Next, bioconversion buffer was added to the cell pellets targeting a cell wet weight of 100 g/L and the cell pellets were resuspended.

To carry out biotransformations, 1 mL of this mixture was transferred into sterile 40 mL amber screw cap vials and 100 μL of an 84:16 mixture of (E)-5-decene:(Z)-5-decene was added along with 100 μL of isopropyl alcohol. This reaction mixture was incubated at 20° C. and 180 rpm for 1 hour before the reaction was quenched via addition of 100 μL of 3M HCL.

To extract the biotransformation products, 5 mL of n-hexane was added. The mixture was thoroughly mixed via shaking in an orbital shaker (20 min @ 250 rpm) and then allowed to settle undisturbed for 20 minutes. An aliquot (1 mL) of the organic layer was analyzed via gas chromatography (GC) using a J&W DB-23 column (30 m×25 mm×25 μm) coupled to an FID detector using the following temperature profile: 45° C. for 0.5 min; ramp 5° C./min to 50° C.; hold 0.5 min; ramp 30° C./min to 220° C.; hold 3.3 min. Retention for substrates and products were verified using authentic standards and were as follows: (E)-5-decene at 2.84 min, (Z)-5-decene at 2.91 min, (E)-5-decen-1-ol at 6.73 min, and (Z)-5-decen-1-ol at 6.79 min.

Results are shown in Table 17. These results demonstrate the capability of members of the CYP153 and AlkB family of enzymes to catalyze the hydroxylation of (E)-5-decene and (Z)-5-decene. Since the (E/Z)-5-decene substrate used in these bioconversion was an 84:16 mixture of the E:Z enantiomers, a product mixture with the same 84:16 ratio of (E)-5-decen-1-ol to (Z)-5-decen-1-ol would be obtained if the terminal hydroxylase exhibits no selectivity for either enantiomer. Our results demonstrate that the terminal hydroxylase does have a preference for one of the two substrate isomers, and isomeric enrichment for a particular alcohol product can be achieved by biohydroxylation.

TABLE 17 Biotransformation rate and selectivity of terminal hydroxylase expressing strains on E/Z-5-decene. (E)-5-decen-1-ol Strain Expressed terminal Terminal hydroxylase formation rate No. hydroxylase Accession No. (mg/L/h) Z/E selectivity SPV001 None n/a 0 n.d. SPV015 CYP153A7 AJ850057 290 ± 63 85 ± 2:15 ± 2 SPV016 CYP153 M. sp. AFO66437 347 73:27 SPV017 CYP153A13N2 AB206793 41 80:20 SPV029 CYP153A13P2 AB206795 91 86:14 SPV0025 CYP153A M. aq. ABM17701 88 80:20 (G307A) SPV0013 CYP153A M. aq. ABM17701 52 77:23 SPV0026 CYP153A M. aq. ABM17701 66 78:22 (G307A) SPV0023 AlkB YP_009076004 244 78:22 SPV0030 AlkB YP_009076004 232 78:22 SPV0031 AlkB YP_009076004 304 79:21 SPV032 AlkB P1 CAB51047 321 77:23 SPV037 AlkB1 AB CAC38027 221 77:23

Example 6 Hydroxylation of 1-dodecene

The purpose of this example is to illustrate the biocatalytic hydroxylation of 1-dodecene to 11-dodecen-1-ol, and the selectivity of the various enzymes towards the production of either 11-dodecen-1-ol or 1,2-epoxydodecane.

The 25 strains detailed in Example 3 were characterized for their ability to convert 1-dodecene to 11-dodecen-1-ol and 1,2-epoxydodecane in whole cell bioconversion reactions.

Overnight cultures of these 25 strains were inoculated from single colonies grown on LB agar plates containing 30 μg/ml of kanamycin into 2.5 mL of LB medium containing 30 μg/ml of kanamycin and incubated for 24 hours at 37° C. and 210 rpm. The overnight culture was used to inoculate 50 or 100 ml of Terrific Broth with a starting OD₆₀₀ of 0.1. After incubation for approximately 2.5 hours at 37° C. and 210 rpm the cultures reached an OD600 of approximately 1.0-1.5, at which point they were induced with 0.5 mM IPTG and supplemented with 0.5 mM 5-aminolevulinic acid, 50 mg/L thiamine, 1.2 mM MgSO₄, and 25 mL of a solution of trace elements (190 mg CaCl₂*2H₂O, 90 mg ZnSO₄*7H₂O, 90 mg CoCl₂*6H₂O, 75 mg CuSO₄*5H₂O, 50 mg MnSO₄*H₂O, 11.1 mg Na_(z)-EDTA*2H₂O and 8.35 mg FeCl₃*6H₂O in 500 ml of ddH₂O). The culture was further incubated for 20 hours at 20° C. and 180 rpm. The cultures were then pelleted via centrifugation at 3900×g at 4° C. for 10 min, washed once with bioconversion buffer (100 mM phosphate buffer (pH7.2), 1% glycerol/0.4% glucose, 100 μg/ml FeSO4*7H2O, and 30 μg/ml kanamycin), and pelleted again. Next, bioconversion buffer was added to the cell pellets targeting a cell wet weight of 100 g/L and the cell pellets were resuspended.

To carry out biotransformations, 1 mL of this mixture was transferred into sterile 40 mL amber screw cap vials and 100 μL of 1-dodecene was added along with 100 μL of isopropyl alcohol. This reaction mixture was incubated at 20° C. and 180 rpm for 4 hour before the reaction was quenched via addition of 100 μL of 3M HCL.

To extract the biotransformation products, 5 mL of n-hexane was added. The mixture was thoroughly mixed via shaking in an orbital shaker (20 min @ 250 rpm) and then allowed to settle undisturbed for 20 minutes. An aliquot (1 mL) of the organic layer was analyzed via gas chromatography (GC) using a J&W DB-23 column (30 m×25 mm×25 μm) coupled to an FID detector using the following temperature profile: 45° C. for 0.5 min; ramp 5° C./min to 50° C.; hold 0.5 min; ramp 30° C./min to 220° C.; hold 3.3 min. Retention for substrates and products were verified using authentic standards and were as follows: 1-dodecene at 4.35 min, 11-dodecen-1-ol at 8.91 min, and 1,2-epoxydodecane at 7.86 min.

Results are shown in Table 18. These results demonstrate the capability of members of the CYP153 and AlkB family of enzymes to catalyze the hydroxylation of 1-dodecene to produce 11-dodecen-1-ol. Furthermore, some of these enzyme are able to selectively form 11-dodecen-1-ol over the energetically favored 1,2-epoxydodecane product.

TABLE 18 Biotransformation rate and selectivity of terminal hydroxylase expressing strains on 1-dodecene. Terminal hydroxylase 11-dodecen-1-ol 11-dodecen-1-ol:1,2- Strain Expressed terminal Accession No. titer epoxydodecane No. hydroxylase (protein ID) (mg/L) selectivity SPV001 None n/a  0 n.d. SPV015 CYP153A7 AJ850057 85 ± 33 86 ± 13:14 ± 13 SPV016 CYP153 M. sp. AFO66437 68 61:39 SPV017 CYP153A13N2 AB206793 25 54:46 SPV028 CYP153A13N3 AB206799 64 55:45 SPV029 CYP153A13P2 AB206795 21 55:45 SPV0025 CYP153A M. aq. ABM17701 52 65:35 (G307A) SPV0026 CYP153A M. aq. ABM17701 42 61:39 (G307A) SPV0023 AlkB YP_009076004 26 67:33 SPV0030 AlkB YP_009076004 34 67:33 SPV0031 AlkB YP_009076004 71 68:32 SPV032 AlkB P1 CAB51047 86 69:31 SPV037 AlkB1 AB CAC38027 38 48:52

Example 7 Hydroxylation of (Z)-5-Hexadecene by Cytochromes P450 of the CYP153 Family

The purpose of this example is to illustrate the biocatalytic hydroxylation of (Z)-5-hexadecene by members of the CYP153 family.

The following strains are constructed by (1) restriction digest of a synthesized DNA fragment containing the target gene insert and the desired expression plasmid followed by (2) ligation of the digested fragment and (3) transformation of the ligation mixture into the desired E. coli strain (Table 19).

TABLE 19 Strains constructed. Strain Hydroxylase No. Plasmid E. coli strain Gene insert Accession No. SPV0001 pET28a(+) E. coli BL21 (DE3) None n/a SPV0012 pET28a(+) E. coli BL21 (DE3) Cyp153A P.sp.-CPR_(BM3) Q11ZY2 SPV0025 pColaDuet-1 E. coli BL21 (DE3) Cyp153A M. aq. (G307A)- A1TY82 CPR_(BM3) SPV0026 pET28a(+) E. coli BL21 (DE3) Cyp153A M. aq. (G307A)- A1TY82 CPR_(BM3)

Cultures of the strains listed in Table 19 are inoculated, cultured, and subjected to a biotransformation procedure as described in Example 5 with the exception that (Z)-5-hexadecene was used as the substrate instead of the 84:16 mixture of (E)-5-decene:(Z)-5-decene. The biotransformation products are extracted and then analyzed via gas chromatography as described in Example 5 to identify products including (Z)-11-hexadecene-1-ol and (Z)-5-hexadecene-1-ol.

Example 8 Hydroxylation of (Z)-5-Hexadecene by Cytochromes P450 of the CYP52 Family

The purpose of this example is to illustrate the biocatalytic hydroxylation of (Z)-5-hexadecene by members of the CYP52 family.

Two P450 cytochromes of the CYP52 family were integrated into the P. pastoris CBS7435 Mut^(S) genome along with their corresponding cytochrome P450 reductases (CPR). Biotransformations were performed with these strains to determine whether these P450s hydroxylate (Z)-5-hexadecene. Strains and oligonucleotides disclosed in this example are listed in Tables 20 and 21.

TABLE 20 Genotypes of strains used in Example 8. Strain No. Genotype SPV048 P. pastoris CBS7435 mut^(S) pPpT4_SmiI_cmRED_cmCYP52A3 SPV051 P. pastoris CBS7435 mut^(S) pPpT4_SmiI_ctRED_ctCYP52A13

TABLE 21 Oligonucleotide sequences used in Example 8. SEQ Primer Sequence Description ID NO. OPV 0042 ATGACGGT CYP52A13 10 TCATGACA forward TCATCGC primer OPV 0043 CTGACATC CYP52A13/A3 11 CTCTTGAG reverse CGGC primer OPV 0044 ATGGCTAT CYP52A3 12 TGAGCAGA forward TTATCGAA primer G

Gene sequences for C. tropicalis CYP52A13 (Accession No. AA073953.1), C. tropicalis CPR (Accession No. P37201.1), C. maltosa CYP52A3 (Accession No. P24458.1), as well as the C. maltosa CPR (Accession No. P50126.1), were ordered as synthetic genes (DNA 2.0, Menlo Park, Calif., USA), and cloned into the pT4_S vector using EcoRI/NotI restriction sites for directional cloning. The plasmid containing the expression cassettes for CYP52A3/CPR and CYP52A13/CPR under the control of an AOX promoter and terminator were linearized using the restriction enzyme SmiI and purified. Next, 500 ng of the linearized DNA sequences for expressing CYP52A3/CPR (SEQ ID NO:13) and CYP52A13/CPR (SEQ ID NO:14) were used to transform P. pastoris CBS7435 Mut^(S). The parent strain and the generation of the pT4 S plasmid used to generate the subsequent constructs are described by Gudiminchi et al. (Biotechnology Journal, 2013, 8(1), 146-52).

Colony PCR of the obtained P. pastoris strains was performed to verify the P450 enzymes CYP52A3 and CYP52A13 were present using the Failsafe™ PCR Kit (EPICENTRE® Biotechnologies, Madison, Wis.; Catalog #FS99060) using Premix D and primers shown in Table 21 according to the manufactures recommendations.

Shake flask cultivations of the strains SPV048 and SPV051 were started from single colonies derived from an YBD agar plate (10 g/L Bacto™ yeast extract, 20 g/L Bacto™ peptone, 20 g/L D (+) glucose, 15 g/L agar) containing 100 mg/L Zeocin™. A volume of 45 mL of BMD1 medium (BMD1 (1 L): 10 g/L D (+) glucose autoclaved, 200 mL 10×PPB (10×PPB: 30.0 g/L K₂HPO₄, 118 g/L KH₂PO₄, pH 6.0, autoclaved), 100 mL 10×YNB (10×YNB: 134 g/L Difco™ yeast nitrogen base without amino acids, autoclaved), 2 mL 500×buffer B (buffer B:10 mg/50 mL d-Biotin, filter sterilized), add autoclaved H₂O to 1 L) was inoculated with a single colony and incubated for approximately 63 h at 28° C. to 30° C. and 130 rpm in a 250 mL baffled Erlenmeyer flask. After the initial 63 h incubation 5 mL of BMM10 medium (BMM10 (1 L): 50 mL methanol, 200 mL 10×PPB (10×PPB: 30.0 g/L K₂HPO₄, 118 g/L KH₂PO₄, pH 6.0, autoclaved), 100 mL 10×YNB (10×YNB: 134 g/L Difco™ yeast nitrogen base without amino acids, autoclaved), 2 mL 500× buffer B (buffer B:10 mg/50 mL d-Biotin, filter sterilized), add autoclaved H₂O to 1 L) was added. The cultivations were incubated for 12 h at 28° C. to 30° C., 130 rpm. After 12 hours incubation 0.4 mL of methanol was added to induce expression of the P450 enzymes and their corresponding CPR's and incubated for 12 h at 28° C. to 30° C., 130 rpm. Thereafter, 0.4 mL of methanol was added every 12 h and incubated at 28° C. to 30° C., 130 rpm. Cells were harvested after induction for approximately 72 h to 80 h and a total cultivation time of approximately 132 h to 143 h.

As control a volume of 45 mL of BMD1 medium (BMD1 (1 L): 10 g/L D (+) glucose autoclaved, 200 mL 10×PPB (10×PPB: 30.0 g/L K₂HPO₄, 118 g/L KH₂PO₄, pH 6.0, autoclaved), 100 mL 10×YNB (10×YNB: 134 g/L Difco™ yeast nitrogen base without amino acids, autoclaved), 2 mL 500× buffer B (buffer B:10 mg/50 mL d-Biotin, filter sterilized), add autoclaved H₂O to 1 L) was inoculated with a single colony of strain SPV051 incubated for approximately 63 h at 28° C. to 30° C. and 130 rpm in a 250 mL baffled Erlenmeyer flask. After the initial 63 h incubation 5 mL of BMM10 medium without methanol (BMM10 without methanol (1 L): 200 mL 10×PPB (10×PPB: 30.0 g/L K₂HPO₄, 118 g/L KH₂PO₄, pH 6.0, autoclaved), 100 mL 10×YNB (10×YNB: 134 g/L Difco™ yeast nitrogen base without amino acids, autoclaved), 2 mL 500× buffer B (buffer B:10 mg/50 mL d-Biotin, filter sterilized), add autoclaved H₂O to 1 L) was added. The cultivations were incubated for additional 60 h to 68 h at 28° C. to 30° C., 130 rpm. Cells were harvested after a total cultivation time of approximately 132 h to 143 h.

Cultivations were harvested in 50 mL Falcon tubes via centrifugation at 3000×rcf for 5 min at 4° C. The supernatant was discarded. The pellet was resuspended in 5 mL 100 mM PPB (mix stock solutions: 80.2 mL of 1M K₂HPO₄ (174.18 g/L) with 19.8 mL of 1M KH₂PO₄ (136.09 g/L) autoclaved, add autoclaved H₂O to 1 L and adjust pH 7.4), containing 20% glycerol, pH 7.4 and centrifuged again at 3000×rcf for 5 min at 4° C. (washing step). The supernatant was discarded and the Falcon tube was carefully patted on a Kimwipe to remove excess buffer. Each pellet was weighed to determine the cell wet weight (cww) of the cultures. The washed pellet was resuspended in bioconversion buffer (100 mM PPB (mix stock solutions: 80.2 mL of 1M K₂HPO₄ (174.18 g/L) with 19.8 mL of 1M KH₂PO₄ (136.09 g/L) autoclaved, add autoclaved H₂O to 1 L and adjust to pH 7.4), 20% glycerol, 0.2% Emulgen 913 (Kao Chemicals, Japan), pH 7.4) targeting a final cell density of ˜200 g cww/L.

1 ml of the resuspended cultivation (200 g cww/L) was dispensed in a 50 mL Falcon tube. 125 μL neat substrate was added to each culture to initiate the bioconversion reactions. The bioconversion reactions were incubated at 30° C. and 200 rpm for 40 h to 48 h. The samples were stored at −80° C. until extraction and analysis of the respective product formation.

250 μL of 3 M HCl was added to each of the frozen samples. After addition of HCl samples were extracted twice with 1×1 mL or 2×2 mL diethyl ether. 10 μL of 10 mg/mL 1-Heptanol or 10 μL of 10 mg/mL 1-Tetradecanol was added to the sample as internal standard. Upon addition of diethyl ether and internal standard the sample was vortexed for 5 min. The entire sample was transferred to new reaction tubes and centrifuged for 10 min/8000×rcf at room temperature. The organic upper phase was transferred to a glass vial and air dried. The sample was resuspended to a final volume of 100 μL to 150 μL using Methyl Tertiary Butyl Ether (MTBE) or resuspended to a final volume of 200 μL using Tetrahydrofuran (THF) and analyzed via gas chromatography (GC).

An Agilent 6890 equipped with an FID detector and a J&W DB-23 column (length: 30 m, I.D. 25 mm, film 25 μm) was used to analyze the samples using the following program: Split ratio of 1:10. 240° C. for the injector inlet: 240° C. for the detector. H₂ at 40.0 mL/min, Air at 450 mL/min, Makeup flow (He) at 45 mL/min. Carrier He at 1.1 mL/min and 13 psi. 45° C. oven for 0.5 min; 5° C./min gradient to 50° C., then hold at 50° C. for 0.5 min; 30° C./min gradient to 220° C., then hold at 220° C. for 333 min. Analysis was performed in triplicate using authentic standards (obtained from Sigma-Aldrich or Bedoukian Research).

Results are shown in Table 22 and FIG. 1. Surprisingly, the CYP52 enzymes exhibit selectivity for one end of the (Z)-5-hexadecene substrate over the other: The SPV048 bioconversion produced 66.8% (Z)-5-hexadecen-1-ol and 33.2% (Z)-11-hexadecen-1-ol while the SPV051 bioconversion produced 27.6% (Z)-5-hexadecen-1-ol and 74.4% (Z)-11-hexadecen-1-ol.

TABLE 22 Results for bioconversions. Induced Selectivity Strain Enzyme (Y/N) Substrate Products [%] SPV048 CYP52A3 N (Z)-5-hexadecene n.d. n/a SPV048 CYP52A3 Y (Z)-5-hexadecene (Z)-5-hexadecen-1-ol 66.8 ± 7.6 (Z)-11-hexadecen-1-ol 33.2 ± 1.0 SPV051 CYP52A13 N (Z)-5-hexadecene n.d. n/a SPV051 CYP52A13 Y (Z)-5-hexadecene (Z)-5-hexadecen-1-ol, 27.6 ± 4.3 (Z)-11-hexadecen-1-ol 74.4 ± 2.2

Example 9 Hydroxylation of 1-dodecyne

The purpose of this example is to illustrate the biocatalytic hydroxylation of 1-dodecyne to 11-dodecyn-1-ol.

The 25 strains detailed in Example 3 were characterized for their ability to convert 1-dodecyne to 11-dodecyn-1-ol in whole cell bioconversion reactions.

Overnight cultures of these 25 strains were inoculated from single colonies grown on LB agar plates containing 30 μg/ml of kanamycin into 2.5 mL of LB medium containing 30 μg/ml of kanamycin and incubated for 24 hours at 37° C. and 210 rpm. The overnight culture was used to inoculate 50 or 100 ml of Terrific Broth with a starting OD₆₀₀ of 0.1. After incubation for approximately 2.5 hours at 37° C. and 210 rpm the cultures reached an OD600 of approximately 1.0-1.5, at which point they were induced with 0.5 mM IPTG and supplemented with 0.5 mM 5-aminolevulinic acid, 50 mg/L thiamine, 1.2 mM MgSO₄, and 25 mL of a solution of trace elements (190 mg CaCl₂*2H₂O, 90 mg ZnSO₄*7H₂O, 90 mg CoCl₂*6H₂O, 75 mg CuSO₄*5H₂O, 50 mg MnSO₄*H₂O, 11.1 mg Na_(z)-EDTA*2H₂O and 8.35 mg FeCl₃*6H₂O in 500 ml of ddH₂O). The culture was further incubated for 20 hours at 20° C. and 180 rpm. The cultures were then pelleted via centrifugation at 3900×g at 4° C. for 10 min, washed once with bioconversion buffer (100 mM phosphate buffer (pH7.2), 1% glycerol/0.4% glucose, 100 μg/ml FeSO4*7H2O, and 30 μg/ml kanamycin), and pelleted again. Next, bioconversion buffer was added to the cell pellets targeting a cell wet weight of 100 g/L and the cell pellets were resuspended.

To carry out biotransformations, 1 mL of this mixture was transferred into sterile 40 mL amber screw cap vials and 100 μL of 1-dodecyne was added along with 100 μL of isopropyl alcohol. This reaction mixture was incubated at 20° C. and 180 rpm for 4 hour before the reaction was quenched via addition of 100 μL of 3M HCL.

To extract the biotransformation products, 5 mL of n-hexane was added. The mixture was thoroughly mixed via shaking in an orbital shaker (20 min @ 250 rpm) and then allowed to settle undisturbed for 20 minutes. An aliquot (1 mL) of the organic layer was analyzed via gas chromatography (GC) using a J&W DB-23 column (30 m×25 mm×25 μm) coupled to an FID detector using the following temperature profile: 45° C. for 0.5 min; ramp 5° C./min to 50° C.; hold 0.5 min; ramp 30° C./min to 220° C.; hold 3.3 min. Retention for substrates and products were verified using authentic standards and were as follows: 1-dodecyne at 6.34 min and 11-dodecyn-1-ol at 9.71 min.

Results are shown in Table 23. These results demonstrate the capability of members of the CYP153 and AlkB family of enzymes to catalyze the hydroxylation of 1-dodecyne to produce 11-dodecyn-1-ol.

TABLE 23 Biotransformation rate and selectivity of terminal hydroxylase expressing strains on 1-dodecyne. Expressed Terminal Strain terminal hydroxylase 11-dodecyn-1-ol titer No. hydroxylase Accession No. (mg/L) SPV001 None n/a 0 SPV015 CYP153A7 AJ850057 67 ± 21 SPV016 CYP153 M. sp. AFO66437 74 SPV029 CYP153A13P2 AB206795 10 SPV0025 CYP153A M. aq. ABM17701 27 (G307A) SPV0013 CYP153A M. aq. ABM17701 48 SPV0026 CYP153A M. aq. ABM17701 49 (G307A) SPV0023 AlkB YP_009076004 150 SPV0030 AlkB YP_009076004 166 SPV0031 AlkB YP_009076004 251 SPV032 AlkB P1 CAB51047 265 SPV037 AlkB1 AB CAC38027 178

Example 10 Hydroxylation of 1-bromodecane

The purpose of this example is to illustrate the biocatalytic hydroxylation of 1-bromodecane to 10-bromodecan-1-ol, and the selectivity of the various enzymes towards 10-bromodecan-1-ol formation over 1-decanal formation as the result of dehalogenation.

The 25 strains detailed in Example 3 were characterized for their ability to convert 1-bromodecane to 10-bromodecan-1-ol in whole cell bioconversion reactions.

Overnight cultures of these 25 strains were inoculated from single colonies grown on LB agar plates containing 30 μg/ml of kanamycin into 2.5 mL of LB medium containing 30 μg/ml of kanamycin and incubated for 24 hours at 37° C. and 210 rpm. The overnight culture was used to inoculate 50 or 100 ml of Terrific Broth with a starting OD₆₀₀ of 0.1. After incubation for approximately 2.5 hours at 37° C. and 210 rpm the cultures reached an OD600 of approximately 1.0-1.5, at which point they were induced with 0.5 mM IPTG and supplemented with 0.5 mM 5-aminolevulinic acid, 50 mg/L thiamine, 1.2 mM MgSO₄, and 25 mL of a solution of trace elements (190 mg CaCl₂*2H₂O, 90 mg ZnSO₄*7H₂O, 90 mg CoCl₂*6H₂O, 75 mg CuSO₄*5H₂O, 50 mg MnSO₄*H₂O, 11.1 mg Na_(z)-EDTA*2H₂O and 8.35 mg FeCl₃*6H₂O in 500 ml of ddH₂O). The culture was further incubated for 20 hours at 20° C. and 180 rpm. The cultures were then pelleted via centrifugation at 3900×g at 4° C. for 10 min, washed once with bioconversion buffer (100 mM phosphate buffer (pH7.2), 1% glycerol/0.4% glucose, 100 μg/ml FeSO4*7H2O, and 30 μg/ml kanamycin), and pelleted again. Next, bioconversion buffer was added to the cell pellets targeting a cell wet weight of 100 g/L and the cell pellets were resuspended.

To carry out biotransformations, 1 mL of this mixture was transferred into sterile 40 mL amber screw cap vials and 100 μL of 1-bromodecane was added along with 100 μL of isopropyl alcohol. This reaction mixture was incubated at 20° C. and 180 rpm for 1 hour before the reaction was quenched via addition of 100 μL of 3M HCL.

To extract the biotransformation products, 5 mL of n-hexane was added. The mixture was thoroughly mixed via shaking in an orbital shaker (20 min @ 250 rpm) and then allowed to settle undisturbed for 20 minutes. An aliquot (1 mL) of the organic layer was analyzed via gas chromatography (GC) using a J&W DB-23 column (30 m×25 mm×25 μm) coupled to an FID detector using the following temperature profile: 45° C. for 0.5 min; ramp 5° C./min to 50° C.; hold 0.5 min; ramp 30° C./min to 220° C.; hold 3.3 min. Retention for substrates and products were verified using authentic standards and were as follows: 1-bromodecane at 7.42 min, 10-bromodecan-1-ol at 11.6 min and 1-decanal at 7.24 min.

Results are shown in Table 24. These results demonstrate the capability of members of the CYP153 and AlkB family of enzymes to catalyze the hydroxylation of 1-bromodecane to 10-bromodecan-1-ol. Furthermore, these results demonstrate selective hydroxylation to from 10-bromodecan-1-ol over the dehalogenated product 1-decanal.

TABLE 24 Biotransformation rate and selectivity of terminal hydroxylase expressing strains on 1-bromodecane. 10-bromodecan- Expressed Terminal 1-ol formation Strain terminal hydroxylase rate 10-bromodecan-1-ol:1- No. hydroxylase Accession No. (mg/L/h) decanal selectivity SPV001 None n/a 0 n.d. SPV015 CYP153A7 AJ850057 470 ± 125 93 ± 3:7 ± 3  SPV016 CYP153 M. sp. AFO66437 170 73:27 SPV0025 CYP153A M. ABM17701 80 82:8  aq. (G307A) SPV0013 CYP153A M. ABM17701 131 93:7  aq. SPV0026 CYP153A M. ABM17701 105 83:17 aq. (G307A) SPV0030 AlkB YP_009076004 92 71:29 SPV0031 AlkB YP_009076004 209 83:17 SPV032 AlkB P1 CAB51047 197 86:14 SPV037 AlkB1 AB CAC38027 161 81:19

Example 11 Synthesis of (E)-5-decen-1-ol is carried out according to Scheme 24

E-5-Decene:

The synthesis of E-5-decene is carried out according to the Example 6 disclosed in the US Patent Application No. 2013/0023665 A1. Briefly, into a 25 mL Schlenk flask, the catalyst (Richard Pederson et al. Adv. Synth. Catal. 2002, 344, 728) (0.0032 mmol) and then 0.4 mL of 1-hexene is added under argon atmosphere. The mixture is stirred at 40° C. for 2 hours. The reaction is quenched by filtration through a plug of silica gel (˜2 cm) packed in a Pasteur pipette using pentane as eluent. The reaction solvent is removed under reduced pressured. The desired product, E-5-decene, is purified by distillation and characterized by GC and NMR.

E-5-Decen-1-ol:

E-5-Decene is then subjected to biohydroxylation according to the process disclosed in Example 6 to generate E-5-decen-1-ol. The product is isolated by extraction of the fermentation broth with organic solvent and purified by distillation.

Example 12 Synthesis of (Z)-3-Hexen-1-ol is Carried Out According to Scheme 25

Z-3-Hexene:

A 5-L jacketed 3-necked flask is equipped with a magnetic stir bar, gas feeding tube and a packed bed column containing a dry ice condenser. Z-selective catalyst (see, Scheme 19a) 2.97 g, 0.0035 mol, 0.023 mol % based on 1-butene added) and toluene (240 g) are added. The flask is sparged with argon for 15 min, while being cooled to 15° C. 1-Butene (841 g, 15.0 mol) is added by bubbling into the toluene solution over 10.5 hours. The rate of addition is such that the reaction temperature remains above 10° C. After 10.5 hours, the packed bed column and the dry ice condenser are replaced with a Friedrich condenser. The Friedrich condenser is circulated with 0° C. coolant. The reaction flask is cooled to 10° C. An argon purge with a flow rate of 1 L/minute is maintained for 12 hours. The metathesis catalyst is removed by the in-situ generation of catalyst-tris(hydroxymethyl)phosphine (THP) complex. Tetrakis(hydroxymethyl)phosphonium chloride (TKC) (80% purity, 20.80 g, 25 equivalent to catalyst) and NaHCO₃ (7.35 g, 25 equivalent to catalyst) are added to the solution. The chiller/heater controlling the jacketed flask is set to 40° C. and stirred for 18 hours. The reaction is cooled to 10° C. and washed with water (500 mL) and brine (500 mL) and dried over anhydrous Na₂SO₄. Z-3-Hexene is isolated by distillation.

Z-3-Hexen-1-ol:

Z-3-Decene is subjected to biohydroxylation according to the process disclosed in Example 6 to generate Z-3-hexen-1-ol. The product is isolated by extraction of the fermentation broth with organic solvent and purified by distillation.

Example 13 Synthesis of (Z)-11-Hexadecenol is Carried Out According to Scheme 26

Z-5-Hexadecene:

The cross metathesis reactions of 1-hexene and dodec-1-ene is carried out in a 250 mL three-necked round-bottomed flask fitted with a condenser, thermometer and septum. The dodec-1-ene (20 mL) is transferred to the reaction flask along with 4 mole equivalent of 1-hexene and the mixture is heated to the desired reaction temperature (ranging from 30 to 100° C.) using an oil bath on a controlled hotplate magnetic stirrer. Thereafter 0.5 mol % of the catalyst (based on dodec-1-ene added; see Scheme 19a) is added to the flask and the reaction mixture is continuously stirred with a magnetic stirrer bar until the formation of the primary metathesis products is completed. The progress of the reaction is monitored by GC/FID. The sample is prepared for GC analysis by diluting an aliquot (0.3 mL) of the sample, taken at various reaction time intervals, with 0.3 mL toluene and quenched with 2 drops of tert-butyl hydrogen peroxide prior to analysis. Once dodec-1-ene is completely consumed, the reaction is quenched with tert-butyl hydrogen peroxide and filtered through a plug of silica using hexane as eluent. The hexane filtrate is concentrated and the Z-5-hexadecene is isolated by distillation.

Z-11-Hexadecen-1-ol:

Z-5-Hexadecene is subjected to biohydroxylation according to the process disclosed in Example 6 to generate Z-11-hexadecen-1-ol. The product is isolated by extraction of the fermentation broth with organic solvent, concentrate and silica-gel chromatography.

Example 14 Synthesis of (Z)-11-Hexadecenol is Carried Out According to Scheme 27

1-Dodecyne:

The synthesis of 1-dodecyne is carried out according to the protocol described in Oprean, Joan et al. Studia Universitatis Babes-Bolyai, Chemia, 2006, 51, 33.

5-Hexadecyne:

To a −78° C. solution of 1-dodecyne (5 mmol) in THF (20 mL), 2.5 M n-BuLi (5 mmol) in hexane is added dropwise via a syringe. A solution of 1-bromobutane (5 mmol) and TBAI (0.2 mmol) dissolve in THF is then dropwise added to the reaction mixture. The reaction mixture is allowed to warm to room temperature and then heat at 70° C. for 24 hours. The reaction is quenched with 5 mL of 1M NH₄Cl and extract with hexanes (3×). The organic fractions are combined, dry with anhydrous MgSO₄ and concentrate under reduced pressure. The resulting residue is purified by silica gel flash chromatography using 60:1/hexane:ethyl acetate as mobile phase. Fractions containing the desired product are pulled and concentrate. 5-Hexadecyne is further purified by distillation.

Z-5-Hexadecene:

With stirring, a mixture of Lindlar's catalyst (40 mg) in pentane (10 mL) is put under a balloon of hydrogen for 90 min at 0° C. Quinoline (1 mg) is then added and the mixture is allowed to stir at 0° C. for another 30 min. A solution of Z-5-hexadecene (55 mg) in 2 mL of pentane is then added to the reaction mixture via a syringe. The reaction is allowed to warm to room temperature and the progress of the reaction is monitored by GC. After 18 hours of reaction time, the reaction mixture is filtered through a No. 4 Whatman filter paper and the filtrate is concentrated under reduced pressure to afford the desired product, Z-5-hexadecene, which can be further purified by distillation.

Z-11-Hexadecen-1-ol:

Z-5-Hexadecene is then subjected to biohydroxylation according to the process disclosed in Example 6 to generate Z-11-hexadecen-1-ol. The product is isolated by extraction of the fermentation broth with ethyl acetate and further purified by distillation.

Example 15 Synthesis of (Z)-11-hexadecenol is carried out according to Scheme 28

1-Hexyne:

The synthesis of 1-hexyne is carried out according to the protocol described in Oprean, Joan et al. Studia Universitatis Babes-Bolyai, Chemia, 2006, 51, 33.

5-Hexadecyne:

To a −78° C. solution of 1-hexyne (5 mmol) in THF (20 mL), 2.5 M n-BuLi (5 mmol) in hexane is added dropwise via a syringe. A solution of 1-bromodecane (5 mmol) and n-Bu₄NI (TBAI) (0.2 mmol) dissolve in THF is then dropwise added to the reaction mixture. The reaction mixture is allowed to warm to room temperature and then heat at 70° C. for 24 hours. The reaction is quenched with 5 mL of 1M NH₄Cl and extract with hexanes (3×). The organic fractions are combined, dry with anhydrous MgSO₄ and concentrate under reduced pressure. The resulting residue is purified by silica gel flash chromatography using 60:1/hexane:ethyl acetate as mobile phase. Fractions containing the desired product are pulled and concentrate.

Z-5-Hexadecene:

With stirring, a mixture of Lindlar's catalyst (40 mg) in pentane (10 mL) is put under a balloon of hydrogen for 90 min at 0° C. Quinoline (1 mg) is then added and the mixture is allowed to stir at 0° C. for another 30 min. A solution of Z-5-hexadecene (55 mg) in 2 mL of pentane is then added to the reaction mixture via a syringe. The reaction is allowed to warm to room temperature and the progress of the reaction is monitored by GC. After 18 hours of reaction time, the reaction mixture is filtered through a No. 4 Whatman filter paper and the filtrate is concentrated under reduced pressure to afford Z-5-hexadecene, which can be further purified by distillation.

Z-11-Hexadecen-1-ol:

Z-5-Hexadecene is then subjected to biohydroxylation according to the process disclosed in Example 6 to generate Z-11-hexadecen-1-ol. The product is isolated by extraction of the fermentation broth with organic solvent and further purified by distillation.

Example 16 Synthesis of (Z)-11-Hexadecenol is Carried Out According to Scheme 29

5-Hexadecyne:

To a −78° C. solution of 1-hexyne (0.383 g, 4.67 mmol) in THF (20 mL), 2.5 M n-BuLi (1.87 mL, 4.67 mmol) in hexane is added dropwise via a syringe. A solution of 1-bromodecane (4.67 mmol) and n-Bu₄NI (TBAI, 57 mg, 0.16 mmol) dissolved in THF is then dropwise added to the reaction mixture. The reaction mixture is allowed to warm to room temperature and then heat at 70° C. for 24 hours. The reaction is quenched with 5 mL of 1M NH₄Cl and extract with hexanes (3×). The organic fractions are combined, dried with anhydrous MgSO₄, and concentrated under reduced pressure. The resulting residue is purified by silica gel flash chromatography using 60:1 hexane:ethyl acetate as the mobile phase. Fractions containing the desired product, 5-hexadecyne, are pooled and concentrated.

Z-5-Hexadecene:

With stirring, a mixture of Lindlar's catalyst (40 mg) in pentane (10 mL) is put under a balloon of hydrogen for 90 min at 0° C. Quinoline (1 mg) is then added and the mixture is allowed to stir at 0° C. for another 30 min. A solution of Z-5-hexadecene (55 mg) in 2 mL of pentane is then added to the reaction mixture via a syringe. The reaction is allowed to warm to room temperature and the progress of the reaction is monitored by GC. After 18 hours of reaction time, the reaction mixture is filtered through a No. 4 Whatman filter paper and the filtrate is concentrated under reduced pressure to afford the desired product, Z-5-hexadecene.

Z-11-Hexadecen-1-ol:

Z-5-Hexadecene is then subjected to biohydroxylation according to the process disclosed in Example 6 to generate Z-11-hexadecen-1-ol. The product is isolated by extraction of the fermentation broth with organic solvent and purified by distillation.

Example 17 Synthesis of (Z)-11-Hexadecenol is Carried Out According to Scheme 30

Z-5-Hexadecene:

Into an oven-dried three-neck RBF, N-amyl triphenylphosphnium bromide (13.98 g, 33.83 mmol) is dissolved in anhydrous toluene (30 mL). The mixture is allowed to stir via a magnetic stir bar at ambient temperature until complete dissolution of the alkyl phosphonium bromide salt is achieved. A solution of 6.57 g of potassium bis(trimethylsilyl)amide (KHMDS) in anhydrous toluene (30 mL) is then dropwise added to the reaction mixture. Upon complete addition of KHMDS solution to the reaction mixture, the reaction solution is allowed to stir for another 15 minutes, and is then cooled to −78° C. in an acetone and dry ice bath.

A solution of undecanal (4.59 mL, 22.28 mmol) in toluene (40 mL) is then dropwise added to the reaction mixture via an addition funnel. The reaction is stirred at −78° C. for 20 minutes, then allowed to warm at room temperature with stirring for another 30 minutes. The reaction is terminated by addition of methanol (40 mL) and then concentrated under reduced pressure. The resulting residue is triturated with hexanes and white precipitate, triphenyl phosphine oxide, is removed by filtration. The process is repeated until triphenyl phosphine oxide is no longer precipitated out of the solution. The remnant triphenyl phosphine oxide is removed by passing the crude reaction product through a short bed of silica using hexane as mobile phase. Z-5-hexadecene is obtained as a colorless oil.

Z-11-hexadecen-1-ol:

Z-5-Hexadecene is subjected to biohydroxylation according to the process disclosed in Example 6 to generate Z-11-hexadecen-1-ol. The product is isolated by extraction of the fermentation broth with organic solvent and purified by distillation.

Example 18 Synthesis of (Z)-11-Hexadecenol is Carried Out According to Scheme 31

1,12-Dodecanediol:

Dodecane is subjected to biohydroxylation according to the process disclosed in Example 6 to generate dodecane-1,12-diol. The product is isolated by extraction of the fermentation broth with organic solvent and purified by distillation.

11-Dodecen-1-ol:

Monodehydration of 1,12-dodecanediol to 11-dodecen-1-ol is carried out according to a vapor-phase catalytic process as described in M. Segawa et al. Journal of Molecular Catalysis A: Chemical 2009, 310, 166. The catalytic reactions are performed in a fixed-bed down-flow reactor with the inside diameter of 17 mm. Prior to the reactions, an In₂O₃ sample (weight, W=0.50 g) is preheated in the reactor in N₂ flow at 500° C. for 1 h. The temperature of catalyst bed set at a prescribed temperature between 300 and 375° C., and 1,12-dodecanediol is then fed through the reactor top at a liquid feed rate of 2.67 mL per hour together with N₂ flow of 30 mL per min. The effluent is collected at −77° C., analyzed by GC. The 11-dodecen-1-ol product is purified by distillation.

Z-11-Hexadecen-1-ol:

The cross metathesis reactions of 1-hexene and 11-dodecen-1-ol is carried out in a 250 mL three-necked round-bottomed flask fitted with a condenser, thermometer and septum. The 11-dodecen-1-ol (20 mL) is transferred to the reaction flask along with 3 mole equivalents of 1-hexene and the mixture is heated to the desired reaction temperature (ranging from 30 to 100° C.) using an oil bath on a controlled hotplate magnetic stirrer. Thereafter 0.5 mol % of the catalyst (based on 11-dodecen-1-ol added) is added to the flask and the reaction mixture is continuously stirred with a magnetic stirrer bar until the formation of the primary metathesis products is completed. The progress of the reaction is monitored by GC/FID. The sample is prepared for GC analysis by diluting an aliquot (0.3 mL) of the sample, taken at various reaction time intervals, with 0.3 mL toluene and quenched with 2 drops of tert-butyl hydrogen peroxide prior to analysis. Once the limiting starting material, 11-dodecen-1-ol, is completely consumed, the reaction is quenched with tert-butyl hydrogen peroxide and filtered through a plug of silica using hexane as eluent. The hexane filtrate is concentrated and the Z-11-hexadecen-1-ol is obtained by distillation.

Example 19 Synthesis of (Z)-11-Hexadecenol is Carried Out According to Scheme 32

11-Dodecen-1-ol:

1-Dodecene is subjected to biohydroxylation according to the process disclosed in Example 6 to generate 11-dodecen-1-ol. The product is isolated by extraction of the fermentation broth with organic solvent and purified by distillation.

Z-11-Hexadecen-1-ol:

The synthesis of Z-11-hexadecen-1-ol is carried out the same way as described in Example 13.

Example 20

Synthesis of (Z)-11-hexadecenal via hydroxylation of 1-halodecane is carried out according to Scheme 33. In particular, Scheme 33 illustrates the synthesis of an important pheromone, (Z)-11-hexadecenal, utilizing biohydroxylation of 1-halodecane. In this process, biohydroxylation of 1-halodecane provides the desired α,ω-halodecanol. Upon protection of the hydroxyl moiety, the molecule can be alkylated with 1-hexyne to provide the C16 internal alkyne, which can be reduced to the cis-alkene via the use of the Lindlar's hydrogenation catalyst. Deprotection of the alcohol moiety, followed by oxidation of the free alcohol to aldehyde provides the desired product, (Z)-11-hexadecenal.

10-Halodecanol:

1-Halodecane is subjected to biohydroxylation according to the process disclosed in Example 6 to generate 11-halodecanol. The product is isolated by extraction of the fermentation broth with organic solvent and purified by distillation.

THP-Protected 10-Halodecanol:

Into an oven-dried 50 mL round-bottom flask, 10-halodecan-1-ol (1 mmol) and p-toluenesulfonic acid monohydrate (catalytic amount) were dissolved in 20 mL of dichloromethane. The mixture was allowed to mix at 0° C. for 10 min. With stirring, 3,4-Dihydro-2H-pyran (5 mmol) dissolved in 10 mL of dichloromethane was then dropwise added to the reaction mixture at 0° C. The progress of the reaction was monitored by TLC and the reaction was observed to be completed within 3 hours. The reaction mixture was concentrated under reduced pressure and the resulting residue was purified by silica gel chromatography. Fractions contain the desired product were pulled and the solvent was removed under reduced pressure to afford the desired product.

THP-Protected 11-Hexadecyn-1-Ol:

To a −78° C. solution of 1-hexyne (5 mmol) in THF (20 mL), 2.5 M n-BuLi (5 mmol) in hexane is added dropwise via a syringe. A solution of THP-protected 10-halodecanol (5 mmol) and n-Bu₄NI (TBAI) (0.16 mmol) dissolve in THF is then dropwise added to the reaction mixture. The reaction mixture is allowed to warm to room temperature and then heat at 70° C. for 24 hours. The reaction is quenched with 5 mL of 1M NH₄Cl and extract with hexanes (3×). The organic fractions are combined, dry with anhydrous MgSO₄ and concentrate under reduced pressure. The resulting residue is purified by silica gel flash chromatography using 60:1/hexane:ethyl acetate as mobile phase to obtain the desired product, THP-protected 11-hexadecyn-1-ol, as pure component.

THP-Protected Z-11-Hexadecenol:

With stirring, a mixture of Lindlar's catalyst (40 mg) in pentane (10 mL) is put under a balloon of hydrogen for 90 min at 0° C. Quinoline (1 mg) is then added and the mixture is allowed to stir at 0° C. for another 30 min. A solution of THP-protected 11-hexadecyn-1-ol (55 mg) in 2 mL of pentane is then added to the reaction mixture via a syringe. The reaction is allowed to warm to room temperature and the progress of the reaction is monitored by GC. After 18 hours of reaction time, the reaction mixture is filtered through a No. 4 Whatman filter paper and the filtrate is concentrated under reduced pressure to afford the desired product.

Z-11-Hexadecenol:

THP-protected Z-11-hexadecenol (1 mmol) is dissolved in methanol (10 mL) with a catalytic amount of monohydrate p-TsOH. The mixture is heated to 70° C. for 30 min. The reaction solvent is removed under reduced pressure and the resulting residue is re-suspended in hexane and purified by silica gel flash chromatography using 9:1/hexane:ethyl acetate as mobile phase. Pure fractions containing the desired product is pulled and concentrated to dryness to provide the desired product, Z-11-hexadecenol.

Z-11-Hexadecenal:

To a 25 mL RBF, Pyridinium chlorochromate (PCC) (90 mg, 0.41 mmol) was dissolved in dichloromethane (15 mL) and the mixture was allowed to stir at ambient temperature for 10 min. A solution of Z-11-Hexadecen-1-ol (48 mg) in dichloromethane (15 ml) was then dropwise added to the reaction mixture. The progress of the oxidation was monitored by TLC and deemed to be finished within 2 hrs. The reaction mixture was filtered through a bed of silica and the filtrate was concentrated under reduced pressure. The resulting residue was purify by silica gel flash chromatography to provide 7 mg of Z-11-hexadecen-1-al, a yield of 14%.

Example 21

Synthesis of Codling Moth pheromone via hydroxylation of 1-bromohexane is carried out according to Scheme 34. Illustrated in Scheme 34 below are possible approaches to the synthesis of Codling Moth pheromone utilizing biohydroxylation of 1-bromohexane and (E,E)-2,4-hexadiene to generate 6-bromo-hexan-1-ol and (E,E)-2,4-hexadien-1-ol, respectively, as key and novel steps. Alternatively, (E,E)-2,4-hexadien-1-ol can be synthesized by reduction of sorbic acid with lithium aluminum hydride. In a similar fashion, 1-bromohexane was subjected to biohydroxylation to generate 1-bromohexanol. With appropriate chemical manipulation, the coupling of (E,E)-2,4-hexadien-1-ol derivative to 1-bromohexanol derivative can be carried out to obtain the desired Codling Moth pheromone as shown in Scheme 34 below.

(E,E)-2,4-Hexadien-1-ol:

(E,E)-2,4-hexadiene is subjected to biohydroxylation according to the process disclosed in Example 6 to generate (E,E)-2,4-hexadienol. The product is isolated by extraction of the fermentation broth with ethyl acetate and purified by distillation.

Alternatively, (E,E)-2,4-hexadienol can also be made by reduction of sorbic acid with one equivalent of lithium aluminium hydride in diethyl ether solvent according to known literature procedures.

6-Bromo-1-Hexanol:

1-Bromohexane is subjected to biohydroxylation according to the process disclosed in Example 6 to generate 6-bromo-1-hexanol. The product is isolated by extraction of the fermentation broth with ethyl acetate and purified by distillation.

THP-Protected 6-Bromo-1-Hexanol:

Into an oven-dried 50 mL round-bottom flask, 6-Bromo-1-hexanol (1 mmol) and p-toluenesulfonic acid monohydrate (catalytic amount) are dissolved in 20 mL of dichloromethane. The mixture was allowed to mix at 0° C. for 10 min. With stirring, 3,4-Dihydro-2H-pyran (3 mmol) dissolved in 10 mL of dichloromethane was then dropwise added to the reaction mixture at 0° C. The progress of the reaction was monitored by TLC and the reaction was observed to be completed within 3 hours. The reaction mixture was concentrated under reduced pressure and the resulting residue was purified by silica gel chromatography. Fractions contain the desired product were pulled and the solvent was removed under reduced pressure to afford the desired product.

1-Bromo-2,3-Hexadiene:

Into an oven-dried 50 mL round-bottom flask, CaH₂ (1 mmol) is suspended in anhydrous THF (10 mL) at −78° C. and to this a solution of 2,4-hexadien-1-ol in 10 mL of THF is then dropwise added. The reaction solution is allowed to stir at −78° C. for 30 min. Phosphorus tribromide (1 mmol) dissolves in THF (10 mL) is then dropwise added to the reaction mixture via an addition funnel. The reaction mixture is allowed to warm to ambient temperature and allows to react until complete consumption of the starting material is achieved that can be monitored by TLC. The reaction mixture is quenched with 5 mL of 1 M NH4Cl and extracted three times with ethyl acetate. The organic fractions are combined, dry with anhydrous MgSO4, and concentrate to dryness. The resulting residue is purified by silica gel chromatography to obtain 1-bromo-2,3-hexadiene as pure compound.

THP-Protected (8E,10E)-Dodecadien-1-ol:

Into an oven-dried 50 mL round-bottom flask, freshly prepared magnesium strip (0.5 g) is added along with a few crystals of iodide and 10 mL of anhydrous THF. A solution of 1-bromo-2,3-hexadiene in THF (10 mL) is then dropwise added to the reaction vessel and a gentle reflux of the reaction mixture is maintained via a heating oil bath. A solution of THP-protected 6-bromo-1-hexanol (1 mmol) in THF (10 mL) is then dropwise added to the reaction mixture via an additional funnel. The reaction mixture is maintained at reflux and the progress of the reaction is monitored by TLC. Once the starting material, 1-bromo-2,3-hexadiene, is observed to have been consumed, the reaction is quenched with 10 mL of water. The reaction mixture is extracted with ethyl acetate (3×) and the organic fractions are combined, dry with anhydrous MgSO₄ and concentrate. THP-protected (8E,10E)-dodecadien-1-ol is further purified by silica gel chromatography.

(8E,10E)-Dodecadien-1-ol:

THP-protected (8E,10E)-dodecadien-1-ol (1 mmol) is dissolved in methanol (10 mL) with a catalytic amount of monohydrate p-toluenesulphonic acid. The mixture is heated to 70° C. for 30 min. The reaction solvent is removed under reduced pressure and the resulting residue is re-suspended in hexane and purified by silica gel flash chromatography using 9:1/hexane:ethyl acetate as mobile phase. Pure fractions containing the desired product are combined and concentrated to dryness to provide the desired product, (8E,10E)-dodecadien-1-ol.

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Although the foregoing has been described in some detail by way of illustration and example for purposes of clarity and understanding, one of skill in the art will appreciate that certain changes and modifications can be practiced within the scope of the appended claims. All publications, patents, patent applications, and sequence accession numbers cited herein are hereby incorporated by reference in their entirety for all purposes.

INFORMAL SEQUENCE LISTING

AlkB construct for DNA synthesis Oligonucleotide Name: 1406_provivi 023 SEQ ID NO: 1 AGCCATCATCATCATCATCACAGCAGC AlkB P1 construct for DNA synthesis Oligonucleotide Name: 1406_provivi 024 SEQ ID NO: 2 CACTATAGGGGAATTGTGAGCGGATAACAATTCC AlkB1A.p. construct for DNA synthesis Oligonucleotide Name: 1406_provivi 025 SEQ ID NO: 3 CTATAGGGGAATTGTGAGCGGATAACAATTCCC AlkB2A.p. construct for DNA synthesis Oligonucleotide Name: 1406_provivi 026 SEQ ID NO: 4 TGGTGGTGCTCGAGTGCGGCCGCAAGCTTCTAAT Fwd primer for alkB constructs Oligonucleotide Name: oPV001 SEQ ID NO: 5 CTTAAATCTCGTAGCGACTAATTTAATAAAAATTG Rev primer for alkB constructs Oligonucleotide Name: oPV002 SEQ ID NO: 6 AAACAGAAGCTTGGCTGCAGGTCG Fwd primer from alkG Oligonucleotide Name: oPV003 SEQ ID NO: 7 CTTATTCCTGAGGATTGGTGCTGCC Rev primer from alkG Oligonucleotide Name: oPV004 SEQ ID NO: 8 GGCAGCACCAATCCTCAGGAATAAG Fwd primer from SfiI Oligonucleotide Name: oPV005 SEQ ID NO: 9 GATGCCGCTGGATCTGGCCTAGA CYP52A13 forward primer Oligonucleotide Name: OPV 0042 SEQ ID NO: 10 ATGACGGTTCATGACATCATCGC CYP52A13/A3 reverse primer Oligonucleotide Name: OPV 0043 SEQ ID NO: 11 CTGACATCCTCTTGAGCGGC CYP52A3 forward primer Oligonucleotide Name: OPV 0044 SEQ ID NO: 12 ATGGCTATTGAGCAGATTATCGAAG DNA sequence of the linearized construct for expressing CYP52A3/CPR used for genome integration SEQ ID NO: 13 AGATCCAATTCCCGCTTTGACTGCCTGAAATCTCCATCGCCTACAATGA TGACATTTGGATTTGGTTGACTCATGTTGGTATTGTGAAATAGACGCAG ATCGGGAACACTGAAAAATACACAGTTATTATTCATTTAAATAACATCC AAAGACGAAAGGTTGAATGAAACCTTTTTGCCATCCGACATCCACAGGT CCATTCTCACACATAAGTGCCAAACGCAACAGGAGGGGATACACTAGCA GCAGACCGTTGCAAACGCAGGACCTCCACTCCTCTTCTCCTCAACACCC ACTTTTGCCATCGAAAAACCAGCCCAGTTATTGGGCTTGATTGGAGCTC GCTCATTCCAATTCCTTCTATTAGGCTACTAACACCATGACTTTATTAG CCTGTCTATCCTGGCCCCCCTGGCGAGGTTCATGTTTGTTTATTTCCGA ATGCAACAAGCTCCGCATTACACCCGAACATCACTCCAGATGAGGGCTT TCTGAGTGTGGGGTCAAATAGTTTCATGTTCCCCAAATGGCCCAAAACT GACAGTTTAAACGCTGTCTTGGAACCTAATATGACAAAAGCGTGATCTC ATCCAAGATGAACTAAGTTTGGTTCGTTGAAATGCTAACGGCCAGTTGG TCAAAAAGAAACTTCCAAAAGTCGGCATACCGTTTGTCTTGTTTGGTAT TGATTGACGAATGCTCAAAAATAATCTCATTAATGCTTAGCGCAGTCTC TCTATCGCTTCTGAACCCCGGTGCACCTGTGCCGAAACGCAAATGGGGA AACACCCGCTTTTTGGATGATTATGCATTGTCTCCACATTGTATGCTTC CAAGATTCTGGTGGGAATACTGCTGATAGCCTAACGTTCATGATCAAAA TTTAACTGTTCTAACCCCTACTTGACAGCAATATATAAACAGAAGGAAG CTGCCCTGTCTTAAACCTTTTTTTTTATCATCATTATTAGCTTACTTTC ATAATTGCGACTGGTTCCAATTGACAAGCTTTTGATTTTAACGACTTTT AACGACAACTTGAGAAGATCAAAAAACAACTAATTATTGAAAGAATTCC GAAACGATGGCTTTGGATAAGTTGGATTTGTACGTCATCATTGTTCTTG CTGTTGCTGTTGCTGCCTACTTTGCTAAAAACCAGTTCTTGGATCAACC TCAGGACACTGGTTTCTTGTCAAACGATACAGCTGGTGGAAATAGTAGA GATATCCTTGAAACTTTGAAGAAAAACAATAAGAACACATTGCTTTTGT TCGGATCTCAAACCGGTACTGCTGAGGACTACGCCAATAAGCTTTCAAG AGAAATCCATAGTAGATTCGGATTGAAAACTATGGTTGCCGATTTCGCA GATTACGACTGGGATAACTTTGGTGACATCCCTAACGATATCTTGGTTT TCTTTATCGTCGCCACCTATGGAGAGGGAGAACCAACTGACAACGCAGA TGAGTTTCACACCTGGTTGACTGACGAAGCTGATACACTTTCCACCTTG AGATACACCGTTTTCGGTTTGGGAAACTCAACTTACGAATTTTACAACG CTATCGGTAGAAAGTTTGACAGACTTTTGGAAGAGAAAGGTGGAGAGAG ATTTGCTGATTATGGTGAAGGAGATGACGGTACAGGAACCCTTGACGAG GATTTCTTGACATGGAAGGACAACGTTTTCGATACCCTTAAAAACGATT TGAACTTCGAAGAGAGAGAGTTGAAGTACGAACCTAACGTTAAGCTTAC AGAAAGAGATGACTTGACCGTTGATGACTCTGAGGTCTCCTTGGGAGAA CCAAATAAGAAATACATCCAATCTGAAGAGATCGACTTGACAAAGGGTC CTTTTGATCATACCCACCCATATCTTGCAAAGATCTCTAAGACTAGAGA GTTGTTTGCTTCCAAGGAAAGAAACTGTGTTCATGTCGAGTTCGATGTT TCTGAATCCAATCTTAAGTACACTACAGGAGACCACTTGGCCGTTTGGC CATCAAACAGTGATGAGAATATTGCAAAGTTCATCAAATGCTTTGGTTT GGATGACAAGATTAACACTGTTTTCGAACTTAAAGCCTTGGATTCTACA TACCAAATTCCATTCCCTAATCCAATCACCTATGGAGCAGTTGTCAGAC ATCACTTGGAAATTTCAGGTCCTGTTAGTAGACAGTTTTTCCTTGCTAT CGCCGGATTCGCTCCAGACGAAGAGACTAAGAAAACTTTTACAAGAATC GGTAACGATAAGCAAGAATTTGCCAACAAGATCACAAGAAAGAAATTGA ACGTTGCAGACGCTCTTTTGTTTGCTTCAAATGGTAGACCTTGGAGTGA TGTTCCATTTGAGTTCATTATCGAAAACGTCCCTCATTTGCAACCAAGA TACTACTCTATCTCTTCCTCAAGTTTGTCCGAGAAGCAGACTATTAATA TCACAGCTGTTGTCGAAGTTGAAGAGGAAGCAGACGGAAGAGCTGTCAC CGGTGTTGTCACTAACCTTTTGAAGAATATTGAGATCGAACAGAACAAG ACTGGAGAAAAACCTGTTGTCCATTACGATTTGTCTGGTCCAAGAAACA AGTTTAACAAGTTTAAGTTGCCTGTTCACGTCAGAAGATCCAACTTTAA GCTTCCTAAAAATACCACTACACCAGTTATTTTGATCGGTCCTGGAACT GGTGTTGCTCCACTTAGAGGTTTCGTCAGAGAGAGAGTTCAACAGGTCA AGAACGGAGTTAACGTCGGTAAAACTGTTTTGTTTTATGGATGTAGAAA CGAACATGATGACTTCTTGTACAAGCAAGAGTGGTCTGAATATGCTTCC GTTTTGGGAGAGAATTTTGAAATGTTCACTGCCTTTTCTAGACAAGACC CATCCAAGAAAGTTTACGTCCAGGATAAGATTGCAGAAAACTCTAAAGT TGTCAACGATCTTTTGAACGAAGGAGCTATCATCTATGTTTGCGGTGAC GCCTCAAGAATGGCAAGAGATGTTCAAAGTACTATTGCTAAGATCGTCG CCAAACACAGAGAGATTCAGGAAGATAAAGCTGTCGAGTTGGTTAAATC CTGGAAAGTTCAGAATAGATATCAAGAAGATGTTTGGTAAGCGGCCGCT CAAGAGGATGTCAGAATGCCATTTGCCTGAGAGATGCAGGCTTCATTTT TGATACTTTTTTATTTGTAACCTATATAGTATAGGATTTTTTTTGTCAT TTTGTTTCTTCTCGTACGAGCTTGCTCCTGATCAGCCTATCTCGCAGCA GATGAATATCTTGTGGTAGGGGTTTGGGAAAATCATTCGAGTTTGATGT TTTTCTTGGTATTTCCCACTCCTCTTCAGAGTACAGAAGATTAAGTGAG ACCTTCGTTTGTGCGGATCCAACATCCAAAGACGAAAGGTTGAATGAAA CCTTTTTGCCATCCGACATCCACAGGTCCATTCTCACACATAAGTGCCA AACGCAACAGGAGGGGATACACTAGCAGCAGACCGTTGCAAACGCAGGA CCTCCACTCCTCTTCTCCTCAACACCCACTTTTGCCATCGAAAAACCAG CCCAGTTATTGGGCTTGATTGGAGCTCGCTCATTCCAATTCCTTCTATT AGGCTACTAACACCATGACTTTATTAGCCTGTCTATCCTGGCCCCCCTG GCGAGGTTCATGTTTGTTTATTTCCGAATGCAACAAGCTCCGCATTACA CCCGAACATCACTCCAGATGAGGGCTTTCTGAGTGTGGGGTCAAATAGT TTCATGTTCCCCAAATGGCCCAAAACTGACAGTTTAAACGCTGTCTTGG AACCTAATATGACAAAAGCGTGATCTCATCCAAGATGAACTAAGTTTGG TTCGTTGAAATGCTAACGGCCAGTTGGTCAAAAAGAAACTTCCAAAAGT CGGCATACCGTTTGTCTTGTTTGGTATTGATTGACGAATGCTCAAAAAT AATCTCATTAATGCTTAGCGCAGTCTCTCTATCGCTTCTGAACCCCGGT GCACCTGTGCCGAAACGCAAATGGGGAAACACCCGCTTTTTGGATGATT ATGCATTGTCTCCACATTGTATGCTTCCAAGATTCTGGTGGGAATACTG CTGATAGCCTAACGTTCATGATCAAAATTTAACTGTTCTAACCCCTACT TGACAGCAATATATAAACAGAAGGAAGCTGCCCTGTCTTAAACCTTTTT TTTTATCATCATTATTAGCTTACTTTCATAATTGCGACTGGTTCCAATT GACAAGCTTTTGATTTTAACGACTTTTAACGACAACTTGAGAAGATCAA AAAACAACTAATTATTGAAAGAATTCCGAAACGATGGCTATTGAGCAGA TTATCGAAGAGGTTTTGCCTTACTTGACTAAATGGTACACTATCCTTTT TGGTGCCGCTTTCACATACTTTTTGTCAATCGCACTTAGAAACAAATAC TACGAGTACAAGTTGAAGTGTGAAAACCCACCTTACTTCAAGACTGCTG GATTCGTTGGTATCCCTGGATTGATTGATGTCATCAAGGCTAAGAACGC TGGTAAATTGGCCGATTACGCAGACCAAACATTTGACGAATACCCTCAT CACAGTTTCTATATGACCGTTGCTGGAATGTTGAAAATTGTTCTTACTG TCGATCCAGAAAACATCAAGGCTGTTCTTGCCACACAGTTTAATGACTT CGCATTGGGTGCTAGACATGCCCACTTTGATCCATTGCTTGGAGACGGT ATTTTCACCTTGGATGGAGAAGGTTGGAAACATTCCAGAGCAATGTTGA GACCTCAATTTGCTAGAGAGCAGATTGCCCATGTTAAGGCATTGGAACC ACACGTTCAAGTCCTTGCCAAGCAGATCAAATTGAACAAGGGAGAGACA TTCGATTTGCAAGAATTGTTTTTCAGATTCACCGTTGACACAGCTACCG AGTTTTTGTTCGGAGAATCAGTTCACAGTCTTTACGATGAGAAATTGGG TGTCCCACCTCCAAACAATATTCCTGGAAGAGAAAACTTTGCTAAGGCC TTCAATACCTCACAACATTATTTGGCTACTAGAACATACAGTCAGATGT TCTATTTCTTGACTAACCCAAAGGAGTTTAGAGACTGCAATGCCAAAGT TCACAAGCTTGCACAATACTTCGTCAATAAGGCATTGGATGCTTCTGAA GACGAGGTTGCTGAGAAGTCCAAAGGTGGATACGTTTTCTTGTATGAAC TTGTCAAACAGACTAGAGATCCTAAGGTTTTGCAAGACCAGTTGCTTAA CATTATGGTCGCTGGTAGAGATACTACAGCCGGATTGCTTTCTTTTGCA ATGTTCGAGCTTGCTAGAAACCCAAAGATCTGGAATAAGTTGAGAGAAG AGATCGAAGTTAATTTTGGACTTGGTGAAGAGGCCAGAGTCGACGAAAT CTCATTCGAGACTTTGAAGAAATGCGAGTACTTGAAGGCAGTTCTTAAC GAAACATTGAGAATGTATCCTAGTGTTCCAGTCAATTTTAGAACCGCTA CTAGAGATACCACTTTGCCTAGAGGTGGTGGTAAAGACGGTACTTCTCC TATTTTCGTTCCAAAGGGATCTTCCGTTGTCTACACAGTCTATAAAACC CATAGATTGGAAGAGTACTATGGTAAAGATGCTTACGAGTTTAGACCTG AGAGATGGTTCGAACCATCCACTAGAAAATTGGGTTGGGCCTATGTTCC TTTTAATGGAGGTCCAAGAATTTGCCTTGGACAACAGTTCGCTTTGACT GAGGCCTCTTACGTTATCACAAGACTTGCTCAAATGTTTGAACACTTGG AGTCCAAGGATGAAACTTATCCTCCAAACAAGTGTATCCATTTGACTAT GAATCACAACGAAGGAGTTTTTATTTCTGCTAAGTAGGCGGCCGCTCAA GAGGATGTCAGAATGCCATTTGCCTGAGAGATGCAGGCTTCATTTTTGA TACTTTTTTATTTGTAACCTATATAGTATAGGATTTTTTTTGTCATTTT GTTTCTTCTCGTACGAGCTTGCTCCTGATCAGCCTATCTCGCAGCAGAT GAATATCTTGTGGTAGGGGTTTGGGAAAATCATTCGAGTTTGATGTTTT TCTTGGTATTTCCCACTCCTCTTCAGAGTACAGAAGATTAAGTGAGACC TTCGTTTGTGCGGATCCTTCAGTAATGTCTTGTTTCTTTTGTTGCAGTG GTGAGCCATTTTGACTTCGTGAAAGTTTCTTTAGAATAGTTGTTTCCAG AGGCCAAACATTCCACCCGTAGTAAAGTGCAAGCGTAGGAAGACCAAGA CTGGCATAAATCAGGTATAAGTGTCGAGCACTGGCAGGTGATCTTCTGA AAGTTTCTACTAGCAGATAAGATCCAGTAGTCATGCATATGGCAACAAT GTACCGTGTGGATCTAAGAACGCGTCCTACTAACCTTCGCATTCGTTGG TCCAGTTTGTTGTTATCGATCAACGTGACAAGGTTGTCGATTCCGCGTA AGCATGCATACCCAAGGACGCCTGTTGCAATTCCAAGTGAGCCAGTTCC AACAATCTTTGTAATATTAGAGCACTTCATTGTGTTGCGCTTGAAAGTA AAATGCGAACAAATTAAGAGATAATCTCGAAACCGCGACTTCAAACGCC AATATGATGTGCGGCACACAATAAGCGTTCATATCCGCTGGGTGACTTT CTCGCTTTAAAAAATTATCCGAAAAAATTTTCTAGAGTGTTGTTACTTT ATACTTCCGGCTCGTATAATACGACAAGGTGTAAGGAGGACTAAACCAT GGCTAAACTCACCTCTGCTGTTCCAGTCCTGACTGCTCGTGATGTTGCT GGTGCTGTTGAGTTCTGGACTGATAGACTCGGTTTCTCCCGTGACTTCG TAGAGGACGACTTTGCCGGTGTTGTACGTGACGACGTTACCCTGTTCAT CTCCGCAGTTCAGGACCAGGTTGTGCCAGACAACACTCTGGCATGGGTA TGGGTTCGTGGTCTGGACGAACTGTACGCTGAGTGGTCTGAGGTCGTGT CTACCAACTTCCGTGATGCATCTGGTCCAGCTATGACCGAGATCGGTGA ACAGCCCTGGGGTCGTGAGTTTGCACTGCGTGATCCAGCTGGTAACTGC GTGCATTTCGTCGCAGAAGAGCAGGACTAACAATTGACACCTTACGATT ATTTAGAGAGTATTTATTAGTTTTATTGTATGTATACGGATGTTTTATT ATCTATTTATGCCCTTATATTCTGTAACTATCCAAAAGTCCTATCTTAT CAAGCCAGCAATCTATGTCCGCGAACGTCAACTAAAAATAAGCTTTTTA TGCTCTTCTCTCTTTTTTTCCCTTCGGTATAATTATACCTTGCATCCAC AGATTCTCCTGCCAAATTTTGCATAATCCTTTACAACATGGCTATATGG GAGCACTTAGCGCCCTCCAAAACCCATATTGCCTACGCATGTATAGGTG TTTTTTCCACAATATTTTCTCTGTGCTCTCTTTTTATTAAAGAGAAGCT CTATATCGGAGAAGCTTCTGTGGCCGTTATATTCGGCCTTATCGTGGGA CCACATTGCCTGAATTGGTTTGCCCCGGAAGATTGGGGAAACTTGGATC TGATTACCTTAGCTGCAGGTACCACTGAGCGTCAGACCCCGTAGAAAAG ATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCT TGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCA AGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAG ATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCA AGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACC AGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCA AGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTT CGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATA CCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAG GCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGA GGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTT TCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGG CGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGG CCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGGTACCC DNA sequence of the linearized construct for expressing CYP52A13/CPR used for genome integration SEQ ID NO: 14 AGATCCAATTCCCGCTTTGACTGCCTGAAATCTCCATCGCCTACAATGA TGACATTTGGATTTGGTTGACTCATGTTGGTATTGTGAAATAGACGCAG ATCGGGAACACTGAAAAATACACAGTTATTATTCATTTAAATAACATCC AAAGACGAAAGGTTGAATGAAACCTTTTTGCCATCCGACATCCACAGGT CCATTCTCACACATAAGTGCCAAACGCAACAGGAGGGGATACACTAGCA GCAGACCGTTGCAAACGCAGGACCTCCACTCCTCTTCTCCTCAACACCC ACTTTTGCCATCGAAAAACCAGCCCAGTTATTGGGCTTGATTGGAGCTC GCTCATTCCAATTCCTTCTATTAGGCTACTAACACCATGACTTTATTAG CCTGTCTATCCTGGCCCCCCTGGCGAGGTTCATGTTTGTTTATTTCCGA ATGCAACAAGCTCCGCATTACACCCGAACATCACTCCAGATGAGGGCTT TCTGAGTGTGGGGTCAAATAGTTTCATGTTCCCCAAATGGCCCAAAACT GACAGTTTAAACGCTGTCTTGGAACCTAATATGACAAAAGCGTGATCTC ATCCAAGATGAACTAAGTTTGGTTCGTTGAAATGCTAACGGCCAGTTGG TCAAAAAGAAACTTCCAAAAGTCGGCATACCGTTTGTCTTGTTTGGTAT TGATTGACGAATGCTCAAAAATAATCTCATTAATGCTTAGCGCAGTCTC TCTATCGCTTCTGAACCCCGGTGCACCTGTGCCGAAACGCAAATGGGGA AACACCCGCTTTTTGGATGATTATGCATTGTCTCCACATTGTATGCTTC CAAGATTCTGGTGGGAATACTGCTGATAGCCTAACGTTCATGATCAAAA TTTAACTGTTCTAACCCCTACTTGACAGCAATATATAAACAGAAGGAAG CTGCCCTGTCTTAAACCTTTTTTTTTATCATCATTATTAGCTTACTTTC ATAATTGCGACTGGTTCCAATTGACAAGCTTTTGATTTTAACGACTTTT AACGACAACTTGAGAAGATCAAAAAACAACTAATTATTGAAAGAATTCA AAACGATGGCACTTGATAAACTAGATTTGTACGTGATTATCACCTTAGT GGTTGCTATCGCTGCCTACTTCGCTAAAAACCAATTTCTGGACCAACAG CAGGACACTGGATTTTTGAATACTGATTCCGGTGATGGTAACTCCAGAG ACATTTTACAAGCACTTAAGAAGAATAACAAAAATACTCTACTGTTATT TGGATCACAAACTGGTACAGCTGAAGATTACGCCAACAAACTGTCCCGT GAATTACATTCGAGGTTTGGATTGAAAACAATGGTTGCAGACTTCGCTG ATTATGACTTCGAGAATTTCGGTGATATTACAGAGGACATTTTGGTCTT TTTCATAGTCGCCACTTATGGTGAAGGTGAACCGACTGATAATGCTGAC GAGTTCCACACTTGGCTGACCGAGGAGGCTGATACTTTGAGTACACTGA AGTACACAGTTTTTGGATTGGGTAATTCTACTTACGAATTTTTCAACGC TATTGGTAGGAAGTTCGACAGATTACTGGGTGAGAAAGGTGGCGACAGA TTTGCTGAATACGGTGAAGGCGACGACGGTACTGGAACTTTGGATGAAG ATTTCCTTGCTTGGAAGGACAACGTCTTTGATTCATTGAAAAATGATTT GAATTTCGAGGAGAAAGAGCTTAAGTATGAACCAAACGTAAAATTGACC GAAAGGGACGATTTGAGCGGTAATGATCCAGATGTATCTCTAGGTGAAC CTAATGTGAAATACATCAAAAGTGAAGGTGTGGACCTTACCAAAGGACC TTTCGACCATACCCATCCCTTTTTGGCAAGGATCGTGAAAACAAAAGAG TTGTTCACAAGCGAAGATAGACACTGTGTTCATGTAGAGTTTGACATAT CCGAATCAAACCTTAAGTACACAACAGGTGACCATCTGGCAATCTGGCC ATCAAATTCTGATGAGAATATCAAGCAATTTGCCAAGTGTTTTGGACTG GAGGATAAGCTCGACACTGTCATTGAATTGAAGGCATTGGACTCAACGT ATTCTATTCCATTTCCAAATCCTATCACCTACGGTGCAGTTATCAGACA CCATCTAGAAATAAGTGGTCCTGTCTCAAGACAATTTTTCCTCTCCATC GCCGGATTTGCTCCAGATGAGGAGACTAAAAAGTCCTTCACTAGAATTG GTGGAGACAAACAGGAGTTCGCCAGCAAGGTAACTAGACGTAAGTTTAA CATCGCTGATGCCCTTTTGTTCGCTTCCAACAATCGTCCGTGGTCTGAC GTTCCATTCGAGTTTCTAATTGAAAATGTTCAACACTTGACACCACGTT ATTACTCTATTTCCTCAAGCTCCCTATCAGAAAAACAGACCATTAATGT TACAGCTGTGGTTGAAGCTGAGGAGGAAGCAGACGGCAGGCCTGTTACG GGAGTTGTGACAAATCTGCTTAAAAACATTGAAATTGAACAAAATAAGA CGGGAGAAACTCCTATGGTTCACTATGACTTGAATGGTCCAAGAGGTAA GTTCTCCAAGTTCAGACTGCCCGTTCACGTTAGAAGATCCAACTTTAAG CTCCCAAAGAACTCGACTACACCCGTCATCTTGATTGGTCCAGGTACAG GTGTTGCCCCTTTGAGAGGATTCGTTAGAGAACGTGTACAGCAAGTGAA AAACGGTGTCAATGTGGGTAAAACGGTTTTGTTTTATGGATGTAGAAAT TCCGAACAAGACTTCCTGTACAAGCAGGAATGGTCTGAGTATGCTTCGG TGCTAGGTGAGAACTTTGAGATGTTCAATGCATTCAGTCGTCAAGACCC TACTAAAAAGGTGTACGTTCAAGATAAAATCTTAGAAAATTCTGCACTT GTAGATGAGTTGCTCTCTTCTGGAGCCATAATCTACGTGTGCGGAGATG CTAGTCGTATGGCAAGAGATGTCCAAGCAGCTATCGCTAAAATCGTCGC TAAGTCACGAGACATCCATGAAGATAAGGCCGCAGAGTTGGTGAAGAGC TGGAAAGTTCAAAATCGTTATCAGGAGGATGTTTGGTAAGCGGCCGCTC AAGAGGATGTCAGAATGCCATTTGCCTGAGAGATGCAGGCTTCATTTTT GATACTTTTTTATTTGTAACCTATATAGTATAGGATTTTTTTTGTCATT TTGTTTCTTCTCGTACGAGCTTGCTCCTGATCAGCCTATCTCGCAGCAG ATGAATATCTTGTGGTAGGGGTTTGGGAAAATCATTCGAGTTTGATGTT TTTCTTGGTATTTCCCACTCCTCTTCAGAGTACAGAAGATTAAGTGAGA CCTTCGTTTGTGCGGATCCAACATCCAAAGACGAAAGGTTGAATGAAAC CTTTTTGCCATCCGACATCCACAGGTCCATTCTCACACATAAGTGCCAA ACGCAACAGGAGGGGATACACTAGCAGCAGACCGTTGCAAACGCAGGAC CTCCACTCCTCTTCTCCTCAACACCCACTTTTGCCATCGAAAAACCAGC CCAGTTATTGGGCTTGATTGGAGCTCGCTCATTCCAATTCCTTCTATTA GGCTACTAACACCATGACTTTATTAGCCTGTCTATCCTGGCCCCCCTGG CGAGGTTCATGTTTGTTTATTTCCGAATGCAACAAGCTCCGCATTACAC CCGAACATCACTCCAGATGAGGGCTTTCTGAGTGTGGGGTCAAATAGTT TCATGTTCCCCAAATGGCCCAAAACTGACAGTTTAAACGCTGTCTTGGA ACCTAATATGACAAAAGCGTGATCTCATCCAAGATGAACTAAGTTTGGT TCGTTGAAATGCTAACGGCCAGTTGGTCAAAAAGAAACTTCCAAAAGTC GGCATACCGTTTGTCTTGTTTGGTATTGATTGACGAATGCTCAAAAATA ATCTCATTAATGCTTAGCGCAGTCTCTCTATCGCTTCTGAACCCCGGTG CACCTGTGCCGAAACGCAAATGGGGAAACACCCGCTTTTTGGATGATTA TGCATTGTCTCCACATTGTATGCTTCCAAGATTCTGGTGGGAATACTGC TGATAGCCTAACGTTCATGATCAAAATTTAACTGTTCTAACCCCTACTT GACAGCAATATATAAACAGAAGGAAGCTGCCCTGTCTTAAACCTTTTTT TTTATCATCATTATTAGCTTACTTTCATAATTGCGACTGGTTCCAATTG ACAAGCTTTTGATTTTAACGACTTTTAACGACAACTTGAGAAGATCAAA AAACAACTAATTATTGAAAGAATTCAAAACGATGACGGTTCATGACATC ATCGCTACTTACTTCACAAAGTGGTACGTTATCGTTCCTTTAGCCCTTA TTGCATACAGAGTGCTGGATTACTTCTATGGTAGGTACTTAATGTATAA GTTGGGTGCAAAACCATTTTTCCAGAAACAGACCGACGGTTGTTTCGGT TTTAAGGCTCCTTTGGAATTGCTTAAAAAGAAATCAGACGGTACTCTGA TCGACTTTACATTGCAAAGGATACACGATCTGGATAGACCTGACATTCC CACTTTTACTTTTCCAGTATTCAGCATTAATCTTGTTAACACTCTCGAA CCAGAGAACATAAAAGCTATTTTGGCAACGCAATTCAATGATTTCTCCT TGGGTACCAGACACTCCCACTTTGCTCCACTCCTCGGTGATGGTATTTT CACACTGGACGGTGCAGGATGGAAGCATTCTAGATCCATGCTAAGGCCA CAATTTGCAAGAGAGCAGATTTCCCATGTGAAGCTGTTGGAGCCACATG TGCAGGTCTTTTTCAAGCACGTCCGTAAGGCTCAAGGAAAAACTTTTGA TATTCAGGAGTTGTTTTTCAGATTGACTGTTGATTCAGCCACCGAATTT TTGTTCGGAGAAAGTGTTGAATCGCTGCGTGACGAATCAATTGGAATGA GCATCAACGCACTTGATTTCGATGGTAAAGCAGGTTTTGCTGATGCTTT TAACTACTCTCAAAACTACCTTGCTTCAAGAGCTGTGATGCAACAACTG TACTGGGTTTTGAATGGTAAAAAGTTTAAGGAATGCAATGCCAAGGTAC ACAAGTTCGCTGACTATTATGTTAACAAAGCTCTTGATCTAACACCTGA GCAATTGGAAAAACAAGACGGCTACGTTTTCCTATATGAGTTGGTTAAA CAAACTAGAGACAAACAAGTTTTACGTGATCAGTTGTTGAATATCATGG TAGCTGGCCGAGATACAACAGCAGGACTGTTGTCGTTCGTCTTTTTCGA ACTGGCCAGAAATCCCGAAGTCACAAACAAACTGAGAGAAGAGATCGAG GACAAGTTTGGTTTAGGTGAGAATGCTAGTGTTGAGGACATCAGCTTTG AATCTTTAAAGTCCTGTGAGTACTTGAAGGCTGTGCTGAATGAAACTTT GCGTTTATATCCATCTGTTCCTCAAAATTTCCGTGTCGCTACCAAAAAT ACGACATTGCCAAGAGGAGGAGGCAAAGACGGTCTGAGTCCTGTACTAG TCAGAAAAGGTCAGACTGTGATCTACGGAGTTTATGCAGCCCATAGAAA TCCTGCCGTATATGGAAAAGATGCTTTGGAGTTTCGTCCGGAGAGATGG TTTGAACCAGAAACCAAAAAGCTCGGATGGGCTTTCCTTCCATTCAATG GTGGTCCCAGGATATGTTTAGGTCAACAATTCGCTTTAACTGAAGCATC CTACGTGACAGTGCGTTTGTTACAAGAGTTTGCACATCTTTCCATGGAC CCAGACACTGAGTATCCTCCTAAAAAGATGTCTCATTTGACTATGTCTT TGTTCGATGGTGCAAACATTGAAATGTATTAAGCGGCCGCTCAAGAGGA TGTCAGAATGCCATTTGCCTGAGAGATGCAGGCTTCATTTTTGATACTT TTTTATTTGTAACCTATATAGTATAGGATTTTTTTTGTCATTTTGTTTC TTCTCGTACGAGCTTGCTCCTGATCAGCCTATCTCGCAGCAGATGAATA TCTTGTGGTAGGGGTTTGGGAAAATCATTCGAGTTTGATGTTTTTCTTG GTATTTCCCACTCCTCTTCAGAGTACAGAAGATTAAGTGAGACCTTCGT TTGTGCGGATCCTTCAGTAATGTCTTGTTTCTTTTGTTGCAGTGGTGAG CCATTTTGACTTCGTGAAAGTTTCTTTAGAATAGTTGTTTCCAGAGGCC AAACATTCCACCCGTAGTAAAGTGCAAGCGTAGGAAGACCAAGACTGGC ATAAATCAGGTATAAGTGTCGAGCACTGGCAGGTGATCTTCTGAAAGTT TCTACTAGCAGATAAGATCCAGTAGTCATGCATATGGCAACAATGTACC GTGTGGATCTAAGAACGCGTCCTACTAACCTTCGCATTCGTTGGTCCAG TTTGTTGTTATCGATCAACGTGACAAGGTTGTCGATTCCGCGTAAGCAT GCATACCCAAGGACGCCTGTTGCAATTCCAAGTGAGCCAGTTCCAACAA TCTTTGTAATATTAGAGCACTTCATTGTGTTGCGCTTGAAAGTAAAATG CGAACAAATTAAGAGATAATCTCGAAACCGCGACTTCAAACGCCAATAT GATGTGCGGCACACAATAAGCGTTCATATCCGCTGGGTGACTTTCTCGC TTTAAAAAATTATCCGAAAAAATTTTCTAGAGTGTTGTTACTTTATACT TCCGGCTCGTATAATACGACAAGGTGTAAGGAGGACTAAACCATGGCTA AACTCACCTCTGCTGTTCCAGTCCTGACTGCTCGTGATGTTGCTGGTGC TGTTGAGTTCTGGACTGATAGACTCGGTTTCTCCCGTGACTTCGTAGAG GACGACTTTGCCGGTGTTGTACGTGACGACGTTACCCTGTTCATCTCCG CAGTTCAGGACCAGGTTGTGCCAGACAACACTCTGGCATGGGTATGGGT TCGTGGTCTGGACGAACTGTACGCTGAGTGGTCTGAGGTCGTGTCTACC AACTTCCGTGATGCATCTGGTCCAGCTATGACCGAGATCGGTGAACAGC CCTGGGGTCGTGAGTTTGCACTGCGTGATCCAGCTGGTAACTGCGTGCA TTTCGTCGCAGAAGAGCAGGACTAACAATTGACACCTTACGATTATTTA GAGAGTATTTATTAGTTTTATTGTATGTATACGGATGTTTTATTATCTA TTTATGCCCTTATATTCTGTAACTATCCAAAAGTCCTATCTTATCAAGC CAGCAATCTATGTCCGCGAACGTCAACTAAAAATAAGCTTTTTATGCTC TTCTCTCTTTTTTTCCCTTCGGTATAATTATACCTTGCATCCACAGATT CTCCTGCCAAATTTTGCATAATCCTTTACAACATGGCTATATGGGAGCA CTTAGCGCCCTCCAAAACCCATATTGCCTACGCATGTATAGGTGTTTTT TCCACAATATTTTCTCTGTGCTCTCTTTTTATTAAAGAGAAGCTCTATA TCGGAGAAGCTTCTGTGGCCGTTATATTCGGCCTTATCGTGGGACCACA TTGCCTGAATTGGTTTGCCCCGGAAGATTGGGGAAACTTGGATCTGATT ACCTTAGCTGCAGGTACCACTGAGCGTCAGACCCCGTAGAAAAGATCAA AGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAA ACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGC TACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACC AAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAAC TCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGG CTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACG ATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGC ACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTAC AGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGA CAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAG CTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCC ACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAG CCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTT TGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGGTACCC 

1. A method for synthesizing an olefinic alcohol product, the method comprising incubating an unsaturated hydrocarbon substrate with an enzyme capable of selectively hydroxylating one terminal carbon of the unsaturated hydrocarbon substrate to form an unsaturated hydrocarbon alcohol.
 2. The method of claim 1, wherein the unsaturated hydrocarbon alcohol is the olefinic alcohol product.
 3. The method of claim 1, further comprising converting the unsaturated hydrocarbon alcohol to the olefinic alcohol product.
 4. The method of claim 1, wherein the unsaturated hydrocarbon substrate is an olefinic substrate.
 5. The method of claim 4, wherein the olefinic substrate is a metathesis product.
 6. The method of claim 5, comprising: a) self-metathesizing a terminal olefin in the presence of a metathesis catalyst to form the metathesis product; and b) incubating the metathesis product with an enzyme capable of selectively hydroxylating one terminal carbon of the metathesis product to form an olefinic alcohol product.
 7. The method of claim 6, wherein the terminal olefin has the formula (CH₂═CH)(CH₂)_(m)H, the metathesis product has the formula H(CH₂)_(m)(CH═CH)(CH₂)_(m)H, the olefinic alcohol product has the formula H(CH₂)_(m)(CH═CH)(CH₂)_(m)OH, and m is selected from an integer between 1 and
 17. 8. The method of claim 7, wherein m is selected from an integer between 1 and
 9. 9. The method of claim 5, comprising: a) cross-metathesizing a first terminal olefin and a second different terminal olefin in the presence of a metathesis catalyst to form the metathesis product; and b) incubating the metathesis product with an enzyme capable of selectively hydroxylating one terminal carbon of the metathesis product to form an olefinic alcohol product.
 10. The method of claim 9, wherein the first terminal olefin has the formula (CH₂═CH)(CH₂)_(m)H, the second different terminal olefin has the formula (CH₂═CH)(CH₂)_(n)H, the metathesis product has the formula H(CH₂)_(m)(CH═CH)(CH₂)_(n)H, the olefinic alcohol product has the formula H(CH₂)_(m)(CH═CH)(CH₂)_(n)OH, and m and n are different integers between 1 and
 17. 11. The method of claim 10, wherein m and n are different integers between 1 and
 9. 12. The method of claim 4, wherein the Z:E isomeric ratio of the olefinic alcohol product is different from the Z:E isomeric ratio of the olefinic substrate.
 13. The method of claim 12, wherein the Z:E isomeric ratio of the olefinic alcohol is at least about 1.25 times greater than the Z:E isomeric ratio of the olefinic substrate.
 14. The method of claim 4, wherein the E:Z isomeric ratio of the olefinic alcohol product is different from the E:Z isomeric ratio of the olefinic substrate.
 15. The method of claim 14, wherein the E:Z isomeric ratio of the olefinic alcohol is at least about 1.25 times greater than the E:Z isomeric ratio of the olefinic substrate.
 16. The method of claim 9, wherein the percent regioselectivity is at least about 60%.
 17. The method of claim 9, wherein the olefinic substrate is an asymmetric olefinic substrate.
 18. The method of claim 4, wherein the olefinic substrate is a first terminal olefin.
 19. The method of claim 18, comprising: a) incubating the first terminal olefin with an enzyme capable of selectively hydroxylating the terminal carbon of the terminal olefin to form an α,ω-alkenol; and b) metathesizing the α,ω-alkenol and a second terminal olefin in the presence of a metathesis catalyst to form the olefinic alcohol product.
 20. The method of claim 18, comprising: a) incubating the first terminal olefin with an enzyme capable of selectively hydroxylating the terminal carbon of the terminal olefin to form an α,ω-alkenol; b) protecting the α,ω-alkenol to form a protected α,ω-alkenol; c) metathesizing the protected α,ω-alkenol and a second terminal olefin in the presence of a metathesis catalyst to form a protected olefinic alcohol product; and d) deprotecting the protected olefinic alcohol product to form the olefinic alcohol product.
 21. The method of claim 20, wherein the α,ω-alkenol is protected via esterification and wherein the protected olefinic alcohol product is deprotected via hydrolysis.
 22. The method of claim 21, wherein the α,ω-alkenol is protected via esterification with an acid selected from the group consisting of formate and acetate.
 23. The method of claim 19, wherein the first terminal olefin has the formula (CH₂═CH)(CH₂)_(m)H, the α,ω-alkenol has the formula (CH₂═CH)(CH₂)_(m)OH, the second terminal olefin has the formula (CH₂═CH)(CH₂)_(n)H, the olefinic alcohol product has the formula H(CH₂)_(n)(CH═CH)(CH₂)_(m)OH, and m and n are each independently selected from an integer between 1 and
 17. 24. The method of claim 23, wherein m and n are each independently selected from an integer between 1 and
 9. 25. The method of claim 19, wherein the enzyme produces an olefinic alcohol product:epoxide product ratio of greater than 1:1.
 26. The method of claim 25, wherein the enzyme produces an olefinic alcohol product:epoxide product ratio of greater than 2:1. 27-32. (canceled)
 33. The method of claim 3, comprising: a) incubating an alkenyl halide according to formula X″

wherein X a halogen, a, c, and e are independently integers from 0 to 15, and b and d are independently integers from 0 to 2, provided that at least one of b and d is other than 0, with an enzyme capable of selectively hydroxylating one terminal carbon of the alkene to form a halogen-substituted unsaturated hydrocarbon alcohol according to formula XI″

b) converting the halogen-substituted unsaturated hydrocarbon alcohol to a protected halogen-substituted unsaturated hydrocarbon alcohol according to formula XII″

wherein R is a protecting group; c) forming a reaction mixture comprising the protected halogen-substituted unsaturated hydrocarbon alcohol and a reactant according to formula XIIa″

wherein Y is a halogen and f is an integer from 0 to 15 under conditions sufficient to form an elongated protected unsaturated hydrocarbon alcohol according to formula XIIb″

and d) converting the elongated protected unsaturated hydrocarbon alcohol to the olefinic alcohol product.
 34. The method of claim 3, comprising: a) incubating an unsaturated halogen-substituted hydrocarbon according to formula X′″

wherein X is a halogen, a, c, and e are independently integers from 0 to 30, and b and d are independently integers from 1 to 2, provided that at least one of b and d is other than 0, with an enzyme capable of selectively hydroxylating one terminal carbon of the halogen-substituted unsaturated hydrocarbon to form a halogen-substituted unsaturated hydrocarbon alcohol according to formula XI′″

b) converting the halogen-substituted unsaturated hydrocarbon alcohol to a protected halogen-substituted unsaturated hydrocarbon alcohol according to formula XII′″

wherein R is a protecting group; c) forming a reaction mixture comprising the protected halogen-substituted unsaturated hydrocarbon alcohol and a reactant according to formula XIIa″′

wherein Y is a halogen and f is an interger from 0 to 15 under conditions sufficient to form an elongated protected unsaturated hydrocarbon alcohol according to formula XIIb″′

and d) converting the elongated protected unsaturated hydrocarbon alcohol to the olefinic alcohol product. 35-52. (canceled)
 53. The method of claim 1, wherein the enzyme is a non-heme diiron monooxygenase, a long-chain alkane hydroxylase, or a cytochrome P450.
 54. The method of claim 53, wherein the non-heme diiron monooxygenase is selected from Table 3 or a variant thereof having at least 90% identity thereto, the long-chain alkane hydroxylase is selected from Table 4 or a variant thereof having at least 90% identity thereto, and the cytochrome P450 is selected from Table 5 or a variant thereof having at least 90% identity thereto. 55-58. (canceled)
 59. The method of claim 53, wherein the cytochrome P450 is a member of the CYP52 or CYP153 family.
 60. The method of claim 1, wherein the olefinic alcohol product is a pheromone.
 61. The method of claim 1, wherein the olefinic alcohol product is selected from Table
 1. 62. A method for synthesizing an olefinic aldehyde product, the method comprising oxidizing the olefinic alcohol product synthesized in accordance with the method of claim 1 to form an olefinic aldehyde product.
 63. The method of claim 62, wherein the olefinic aldehyde product is a pheromone.
 64. The method of claim 62, wherein the olefinic aldehyde product is selected from Table
 1. 65. A method for synthesizing an olefinic ester product, the method comprising esterifying the olefinic alcohol product synthesized in accordance with the method of claim 1 to form an olefinic ester product.
 66. The method of claim 65, wherein the olefinic ester product is an acetate ester.
 67. The method of claim 65, wherein the olefinic ester product is a pheromone.
 68. The method of claim 65, wherein the olefinic ester product is selected from Table
 1. 69. A method for synthesizing an olefinic acid product, the method comprising oxidizing the olefinic alcohol product synthesized in accordance with the method of claim 1 to form an olefinic acid product. 70-73. (canceled)
 74. The method of claim 1, wherein the enzyme capable of selectively hydroxylating one terminal carbon of the unsaturated hydrocarbon substrate is active in a whole cell. 